Transgenic GPCR expressing animals

ABSTRACT

The invention provides isolated nucleic acids molecules, designated muscarinic acetylcholine receptor 6 (“mACHR-6”) nucleic acid molecules, which encode polypeptides involved in the modulation of acetylcholine responses in acetylcholine responsive cells. The invention also provides antisense nucleic acid molecules, expression vectors containing mACHR-6 nucleic acid molecules, host cells into which the expression vectors have been introduced, and non-human transgenic animals in which an mACHR-6 gene has been introduced or disrupted. The invention still further provides isolated mACHR-6 polypeptides, fusion polypeptides, antigenic peptides, and anti-mACHR-6 antibodies. Diagnostic, screening, and therapeutic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/282,958, filed on Oct. 28, 2002, now pending, which is acontinuation application of Ser. No. 09/349,755, filed on Jul. 8, 1999,now pending, which is a divisional application of Ser. No. 09/042,780,filed on Mar. 17, 1998, which is a continuation-in-part application ofU.S. Ser. No. 08/985,090, filed Dec. 4, 1997, which issued as U.S. Pat.No. 5,882,893. The contents of this co-pending patent application areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Muscarinic receptors, so named because the actions of acetylcholine onsuch receptors are similar to those produced by the mushroom alkaloidmuscarine, mediate most of the inhibitory and excitatory effects of theneurotransmitter acetylcholine in the heart, smooth muscle, glands andin neurons (both presynaptic and postsynaptic) in the autonomic and thecentral nervous system (Eglen, R. and Watson, N. (1996) Pharmacology &Toxicology 78:59-68). The muscarinic receptors belong to the Gprotein-coupled receptor superfamily (Wess, J. et al. (1990)Comprehensive Medicinal Chemistry 3:423-491). Like all other Gprotein-coupled receptors, the muscarinic receptors are predicted toconform to a generic protein fold consisting of seven hydrophobictransmembrane helices joined by alternative intracellular andextracellular loops, an extracellular amino-terminal domain, and acytoplasmic carboxyl-terminal domain. The mammalian muscarinic receptorsdisplay a high degree of sequence identity, particularly in thetransmembrane domains, sharing approximately 145 invariant amino acids(Wess, J. (1993) TIPS 14:308-313). Moreover, all of the mammalianmuscarinic receptors contain a very large third cytoplasmic loop which,except for the membrane-proximal portions, displays virtually nosequence identity among the different family members (Bonner, T. I.(1989) Trends Neurosci. 12:148-151). Ligand binding to the receptor isbelieved to trigger conformational changes within the helical bundle,which are then transmitted to the cytoplasmic domain, where theinteraction with specific G proteins occurs.

Molecular cloning studies have revealed the existence of fivemolecularly distinct mammalian muscarinic receptor proteins, termed theM₁-M₅ receptors (Bonner, T. I. (1989) Trends Neurosci. 12:148-151; andHulme, E. C. et al. (1990) Annu. Rev. Pharmacol Toxicol. 30:633-673).The M₁ receptor is expressed primarily in the brain (cerebral cortex,olfactory bulb, olfactory tubercle, basal forebrain/septum, amygdala,and hippocampus) and in exocrine glands (Buckley, N. J. et al. (1988) J.Neurosci. 8:4646-4652). The M₂ receptor is expressed in the brain(olfactory bulb, basal forebrain/septum, thalamus and amygdala), and inthe ileum and the heart. The M₃ receptor is expressed in the brain(cerebral cortex, olfactory tubercle, thalamus and hippocampus) thelung, the ileum, and in exocrine glands. The M₄ receptor is expressed inthe brain (olfactory bulb, olfactory tubercle, hippocampus and striatum)and in the lung. Finally, the M₅ receptor is expressed primarily in thebrain (substantia nigra) (Hulme, E. C. et al. (1990) A. Rev. Pharmac.Toxic. 30:633-673).

The two enzymes with which muscarinic receptors interact most directlyare adenylate cyclase and phospholipase C. Studies with cloned receptorshave shown that the M₁, M₃, and M₅ muscarinic receptors are coupled tothe types of G proteins known as Go (a stimulatory protein linked tophospholipase C) or Gq and that their activation results in theactivation of phospholipase C. The M₂ and M₄ muscarinic receptors arecoupled to a Gi protein (an inhibitory protein linked to adenylatecyclase), and their activation results in the inhibition of adenylatecyclase. Through these signal transduction pathways, the muscarinicreceptors are responsible for a variety of physiological functionsincluding the regulation of neurotransmitter release (includingacetylcholine release) from the brain, the regulation of digestiveenzyme and insulin secretion in the pancreas, the regulation of amylasesecretion by the parotid gland, and the regulation of contraction incardiac and smooth muscle (Caulfield, M. P. (1993) Pharmac. Ther.58:319-379).

SUMMARY OF THE INVENTION

This invention provides a novel nucleic acid molecule which encodes apolypeptide, referred to herein as muscarinic acetylcholine receptor 6(“mACHR-6”) polypeptide or protein, which is capable of, for example,modulating the effects of acetylcholine on acetylcholine responsivecells e.g., by modulating phospholipase C signaling/activity. Nucleicacid molecules encoding an mACHR-6 polypeptide are referred to herein asmACHR-6 nucleic acid molecules. In a preferred embodiment, the mACHR-6polypeptide interacts with (e.g., binds to) a protein which is a memberof the G family of proteins. Examples of such proteins include Go, Gi,Gs, Gq and Gt. These proteins are described in Lodish H. et al.Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y.,1995); Dolphin A. C. et al. (1987) Trends Neurosci. 10:53; and BimbaumerL. et al. (1992) Cell 71:1069, the contents of which are expresslyincorporated herein by reference.

In a preferred embodiment, the mACHR-6 polypeptide interacts with (e.g.,binds to) acetylcholine. Acetylcholine is the predominantneurotransmitter in the sympathetic and parasympathetic preganglionicsynapses, as well as in the parasympathetic postganglionic synapses andin some sympathetic postganglionic synapses. Synapses in whichacetylcholine is the neurotransmitter are called cholinergic synapses.Acetylcholine acts to regulate smooth muscle contraction, heart rate,glandular function such as gastric acid secretion, and neural functionsuch as release of neurotransmitters from the brain. The mACHR-6polypeptide of the present invention binds to acetylcholine and servesto mediate the acetylcholine induced signal to the cell. Thus, mACHR-6molecules can be used as targets to modulate acetylcholine inducedfunctions and thus to treat disorders associated with, for example,abnormal acetylcholine levels, or abnormal or aberrant mACHR-6polypeptide activity or nucleic acid expression.

Accordingly, one aspect of the invention pertains to isolated nucleicacid molecules (e.g., cDNAs) comprising a nucleotide sequence encodingan mACHR-6 polypeptide or biologically active portions thereof, as wellas nucleic acid fragments suitable as primers or hybridization probesfor the detection of mACHR-6-encoding nucleic acid (e.g., mRNA). Inparticularly preferred embodiments, the isolated nucleic acid moleculecomprises the nucleotide sequence of SEQ ID NO: 1, 4, or 31, thenucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______, or the coding region or a complementof either of these nucleotide sequences. In other particularly preferredembodiments, the isolated nucleic acid molecule of the inventioncomprises a nucleotide sequence which encodes naturally occurringallelic variants, genetically altered variants and non-human and non-rathomologues of the mACHR-6 polypeptides described herein. Such nucleicacid molecules are identifiable as being able to hybridize to or whichare at least about 30-35%, preferably at least about 40-45%, morepreferably at least about 50-55%, even more preferably at least about60-65%, yet more preferably at least about 70-75%, still more preferablyat least about 80-85%, and most preferably at least about 90-95% or morehomologous to the nucleotide sequence shown in SEQ ID NO: 1, 4, or 31,the nucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______, or a portion of either of thesenucleotide sequences. In other preferred embodiments, the isolatednucleic acid molecule encodes the amino acid sequence of SEQ ID NO:2, 5,or 32 or an amino acid sequence encoded by the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC® as Accession Number______. The preferred mACHR-6 polypeptides of the present invention alsopreferably possess at least one of the mACHR-6 activities describedherein.

In another embodiment, the isolated nucleic acid molecule encodes apolypeptide or portion thereof wherein the polypeptide or portionthereof includes an amino acid sequence which is sufficiently homologousto an amino acid sequence of SEQ ID NO:2, 5, or 32, e.g., sufficientlyhomologous to an amino acid sequence of SEQ ID NO:2, 5, or 32 such thatthe polypeptide or portion thereof maintains an mACHR-6 activity.Preferably, the polypeptide or portion thereof encoded by the nucleicacid molecule maintains the ability to modulate an acetylcholineresponse in an acetylcholine responsive cell. In one embodiment, thepolypeptide encoded by the nucleic acid molecule is at least about30-35%, preferably at least about 40-45%, more preferably at least about50-55%, even more preferably at least about 60-65%, yet more preferablyat least about 70-75%, still more preferably at least about 80-85%, andmost preferably at least about 90-95% or more homologous to the aminoacid sequence of SEQ ID NO:2, 5, or 32 (e.g., the entire amino acidsequence of SEQ ID NO:2, 5, or 32) or the amino acid sequence encoded bythe nucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______. In another preferred embodiment thenucleic acid molecule encodes a polypeptide fragment comprising at least15 contiguous amino acids of SEQ ID NO:2, 5, or 32. In yet anotherpreferred embodiment, the polypeptide is a full length human polypeptidewhich is substantially homologous to the entire amino acid sequence ofSEQ ID NO:2, 5, or 32 (encoded by the open reading frame shown in SEQ IDNO:3, 6, or 33, respectively). In still another preferred embodiment,the nucleic acid molecule encodes a naturally occurring allelic variantof the polypeptide of SEQ ID NO:2, 5, or 32 and hybridizes understringent conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1, 4, or 31, respectively.

In yet another embodiment, the isolated nucleic acid molecule is derivedfrom a human and encodes a portion of a polypeptide which includes atransmembrane domain. Preferably, the transmembrane domain encoded bythe human nucleic acid molecule is at least about 50-55%, preferably atleast about 60-65%, more preferably at least about 70-75%, even morepreferably at least about 80-85%, and most preferably at least about90-95% or more homologous to any of the human transmembrane domains(i.e., amino acid residues 34-59, 109-130, 152-174, 197-219, or 396-416)of SEQ ID NO:2 which are shown as separate sequences designated SEQ IDNOs:7, 9, 10, 11, and 13, respectively, or to any of the rattransmembrane domains (i.e., amino acid residues 34-59, 73-91, 109-130,152-174, 197-219, 360-380, or 396-416 of SEQ ID NO:5 which are shown asseparate sequences designated SEQ ID NOs:14, 15, 16, 17, 18, 19, and 20,respectively or amino acid residues 1-8, 26-47, 69-91, 114-136, 277-297,or 313-333 of SEQ ID NO:32 which are shown as separate sequencesdesignated SEQ ID NOs:34, 35, 36, 37, 38, or 39, respectively). Morepreferably, the transmembrane domain encoded by the human nucleic acidmolecule is at least about 75-80%, preferably at least about 80-85%,more preferably at least about 85-90%, and most preferably at leastabout 90-95% or more homologous to the transmembrane domain (i.e., aminoacid residues 360-380) of SEQ ID NO:2 which is shown as a separatesequence designated SEQ ID NO:12, or at least about 80-85%, morepreferably at least about 85-90%, and most preferably at least about90-95% or more homologous to the transmembrane domain (i.e., amino acidresidues 73-91) of SEQ ID NO:2 which is shown as a separate sequencedesignated SEQ ID NO:8.

In another preferred embodiment, the isolated nucleic acid molecule isderived from a human and encodes a polypeptide (e.g., an mACHR-6 fusionpolypeptide such as an mACHR-6 polypeptide fused with a heterologouspolypeptide) which includes a transmembrane domain which is at leastabout 75% or more homologous to SEQ ID NO:7-13, or to the correspondingrat sequences shown as SEQ ID NOs:14-20 and has one or more of thefollowing mACHR-6 activities: 1) it can interact with (e.g., bind to)acetylcholine; 2) it can interact with (e.g., bind to) a G protein oranother protein which naturally binds to mACHR-6; 3) it can modulate theactivity of an ion channel (e.g., a potassium channel or a calciumchannel); 4) it can modulate cytosolic ion, e.g., calcium,concentration; 5) it can modulate the release of a neurotransmitter,e.g., acetylcholine, from a neuron, e.g., a presynaptic neuron; 6) itcan modulate an acetylcholine response in an acetylcholine responsivecell (e.g., a smooth muscle cell or a gland cell) to, for example,beneficially affect the acetylcholine responsive cell, e.g., a neuron;7) it can signal ligand binding via phosphatidylinositol turnover; and8) it can modulate, e.g., activate or inhibit, phospholipase C activity.

In another embodiment, the isolated nucleic acid molecule is at least 15nucleotides, e.g., at least 15 contiguous nucleotides, in length andhybridizes under stringent conditions to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, 4, or 31 or thenucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______. Preferably, the isolated nucleic acidmolecule corresponds to a naturally-occurring nucleic acid molecule.More preferably, the isolated nucleic acid encodes naturally-occurringhuman mACHR-6 or a biologically active portion thereof. Moreover, giventhe disclosure herein of an mACHR-6-encoding cDNA sequence (e.g., SEQ IDNO:1, 4, or 31), antisense nucleic acid molecules (e.g., molecules whichare complementary to the coding strand of the mACHR-6 cDNA sequence) arealso provided by the invention.

Another aspect of the invention pertains to vectors, e.g., recombinantexpression vectors, containing the nucleic acid molecules of theinvention and host cells into which such vectors have been introduced.In one embodiment, such a host cell is used to produce an mACHR-6polypeptide by culturing the host cell in a suitable medium. If desired,the mACHR-6 polypeptide can then be isolated from the medium or the hostcell.

Yet another aspect of the invention pertains to transgenic non-humananimals in which an mACHR-6 gene has been introduced or altered. In oneembodiment, the genome of the non-human animal has been altered byintroduction of a nucleic acid molecule of the invention encodingmACHR-6 as a transgene. In another embodiment, an endogenous mACHR-6gene within the genome of the non-human animal has been altered, e.g.,functionally disrupted, by homologous recombination.

Still another aspect of the invention pertains to an isolated mACHR-6polypeptide or a portion, e.g., a biologically active portion, thereof.In a preferred embodiment, the isolated mACHR-6 polypeptide or portionthereof can modulate an acetylcholine response in an acetylcholineresponsive cell. In another preferred embodiment, the isolated mACHR-6polypeptide or portion thereof is sufficiently homologous to an aminoacid sequence of SEQ ID NO:2, 5, or 32 such that the polypeptide orportion thereof maintains the ability to modulate an acetylcholineresponse in an acetylcholine responsive cell.

In one embodiment, the biologically active portion of the mACHR-6polypeptide includes a domain or motif, preferably a domain or motifwhich has an mACHR-6 activity. The domain can be transmembrane domain.If the active portion of the polypeptide which comprises thetransmembrane domain is isolated or derived from a human, it ispreferred that the transmembrane domain be at least about 75-80%,preferably at least about 80-85%, more preferably at least about 85-90%,and most preferably at least about 90-95% or more homologous to SEQ IDNO:7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 34, 35, 36, 37,38, or 39. Preferably, the biologically active portion of the mACHR-6polypeptide which includes a transmembrane domain also has one of thefollowing mACHR-6 activities: 1) it can interact with (e.g., bind to)acetylcholine; 2) it can interact with (e.g., bind to) a G protein oranother protein which naturally binds to mACHR-6; 3) it can modulate theactivity of an ion channel (e.g., a potassium channel or a calciumchannel); 4) it can modulate cytosolic ion, e.g., calcium,concentration; 5) it can modulate the release of a neurotransmitter,e.g., acetylcholine, from a neuron, e.g., a presynaptic neuron; 6) itcan modulate an acetylcholine response in an acetylcholine responsivecell (e.g., a smooth muscle cell or a gland cell) to, for example,beneficially affect the acetylcholine responsive cell, e.g., a neuron;7) it can signal ligand binding via phosphatidylinositol turnover; and8) it can modulate, e.g., activate or inhibit, phospholipase C activity.

The invention also provides an isolated preparation of an mACHR-6polypeptide. In preferred embodiments, the mACHR-6 polypeptide comprisesthe amino acid sequence of SEQ ID NO:2, 5, or 32 or an amino acidsequence encoded by the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC® as Accession Number ______. In anotherpreferred embodiment, the invention pertains to an isolated full lengthpolypeptide which is substantially homologous to the entire amino acidsequence of SEQ ID NO:2, 5, or 32 (encoded by the open reading frameshown in SEQ ID NO:3, 6, or 33, respectively) such as a naturallyoccurring allelic variant of the mACHR-6 polypeptides described herein.In yet another embodiment, the polypeptide is at least about 30-35%,preferably at least about 40-45%, more preferably at least about 50-55%,even more preferably at least about 60-65%, yet more preferably at leastabout 70-75%, still more preferably at least about 80-85%, and mostpreferably at least about 90-95% or more homologous to the entire aminoacid sequence of SEQ ID NO:2, 5, or 32 such as a non-human or non-rathomologue of the mACHR-6 polypeptides described herein. In otherembodiments, the isolated mACHR-6 polypeptide comprises an amino acidsequence which is at least about 30-40% or more homologous to the aminoacid sequence of SEQ ID NO:2, 5, or 32 and has an one or more of thefollowing mACHR-6 activities: 1) it can interact with (e.g., bind to)acetylcholine; 2) it can interact with (e.g., bind to) a G protein oranother protein which naturally binds to mACHR-6; 3) it can modulate theactivity of an ion channel (e.g., a potassium channel or a calciumchannel); 4) it can modulate cytosolic ion, e.g., calcium,concentration; 5) it can modulate the release of a neurotransmitter,e.g., acetylcholine, from a neuron, e.g., a presynaptic neuron; 6) itcan modulate an acetylcholine response in an acetylcholine responsivecell (e.g., a smooth muscle cell or a gland cell) to, for example,beneficially affect the acetylcholine responsive cell, e.g., a neuron;7) it can signal ligand binding via phosphatidylinositol turnover; and8) it can modulate, e.g., activate or inhibit, phospholipase C activity.

Alternatively, the isolated mACHR-6 polypeptide can comprise an aminoacid sequence which is encoded by a nucleotide sequence whichhybridizes, e.g., hybridizes under stringent conditions, or is at leastabout 30-35%, preferably at least about 40-45%, more preferably at leastabout 50-55%, even more preferably at least about 60-65%, yet morepreferably at least about 70-75%, still more preferably at least about80-85%, and most preferably at least about 90-95% or more homologous tothe nucleotide sequence of SEQ ID NO:1, 4, or 31 or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC® asAccession Number ______, such as the allelic variants and non-human andnon-rat homologues of the mACHR-6 polypeptides described herein as wellas genetically altered variants generated by recombinant DNAmethodologies. It is also preferred that the preferred forms of mACHR-6also have one or more of the mACHR-6 activities described herein.

The mACHR-6 polypeptide (or protein) or a biologically active portionthereof can be operatively linked to a non-mACHR-6 polypeptide (e.g., apolypeptide comprising heterologous amino acid sequences) to form afusion polypeptide. In addition, the mACHR-6 polypeptide or abiologically active portion thereof can be incorporated into apharmaceutical composition comprising the polypeptide and apharmaceutically acceptable carrier.

The mACHR-6 polypeptide of the invention, or portions or fragmentsthereof, can be used to prepare anti-mACHR-6 antibodies. Accordingly,the invention also provides an antigenic peptide of mACHR-6 whichcomprises at least 8 amino acid residues of the amino acid sequenceshown in SEQ ID NO:2, 5, or 32 and encompasses an epitope of mACHR-6such that an antibody raised against the peptide forms a specific immunecomplex with mACHR-6. Preferably, the antigenic peptide comprises atleast 10 amino acid residues, more preferably at least 15 amino acidresidues, even more preferably at least 20 amino acid residues, and mostpreferably at least 30 amino acid residues. The invention furtherprovides an antibody that specifically binds mACHR-6. In one embodiment,the antibody is monoclonal. In another embodiment, the antibody iscoupled to a detectable substance. In yet another embodiment, theantibody is incorporated into a pharmaceutical composition comprisingthe antibody and a pharmaceutically acceptable carrier.

Another aspect of the invention pertains to methods for modulating acell activity mediated by mACHR-6, e.g., biological processes mediatedby phosphatidylinositol turnover and signaling; secretion of a molecule,e.g., a neurotransmitter from a brain cell, or an enzyme from a glandcell; or contraction of a smooth muscle cell, e.g., an ileum smoothmuscle cell or a cardiac cell, e.g., a cardiomyocyte. Such methodsinclude contacting the cell with an agent which modulates mACHR-6polypeptide activity or mACHR-6 nucleic acid expression such that anmACHR-6-mediated cell activity is altered relative to the same cellularactivity which occurs in the absence of the agent. In a preferredembodiment, the cell (e.g., a smooth muscle cell or a neural cell) iscapable of responding to acetylcholine through a signaling pathwayinvolving an mACHR-6 polypeptide. The agent which modulates mACHR-6activity can be an agent which stimulates mACHR-6 polypeptide activityor mACHR-6 nucleic acid expression. Examples of agents which stimulatemACHR-6 polypeptide activity or mACHR-6 nucleic acid expression includesmall molecules, active mACHR-6 polypeptides, and nucleic acids encodingmACHR-6 that have been introduced into the cell. Examples of agentswhich inhibit mACHR-6 activity or expression include small molecules,antisense mACHR-6 nucleic acid molecules, and antibodies thatspecifically bind to mACHR-6. In a preferred embodiment, the cell ispresent within a subject and the agent is administered to the subject.

The present invention also pertains to methods for treating subjectshaving various disorders, e.g., disorders mediated by abnormal mACHR-6polypeptide activity, such as conditions caused by over, under, orinappropriate expression of mACHR-6. For example, the invention pertainsto methods for treating a subject having a disorder characterized byaberrant mACHR-6 polypeptide activity or nucleic acid expression such asa nervous system disorder, e.g., a cognitive disorder, a sleep disorder,a movement disorder, a schizo-effective disorder, a disorder affectingpain generation mechanisms, a drinking disorder, or an eating disorder;a smooth muscle related disorder, e.g., irritable bowel syndrome, acardiac muscle related disorder, e.g., bradycardia, or a gland relateddisorder, e.g., xerostomia. These methods include administering to thesubject an mACHR-6 modulator (e.g., a small molecule) such thattreatment of the subject occurs.

In other embodiments, the invention pertains to methods for treating asubject having a disorder mediated by abnormal mACHR-6 polypeptideactivity, such as conditions caused by over, under, or inappropriateexpression of mACHR-6, e.g., a nervous system disorder, e.g., acognitive disorder, a sleep disorder, a movement disorder, aschizo-effective disorder, a disorder affecting pain generationmechanisms, a drinking disorder, or an eating disorder; a smooth musclerelated disorder, e.g., irritable bowel syndrome; a cardiac musclerelated disorder, e.g., bradycardia; or a gland related disorder, e.g.,xerostomia. The method includes administering to the subject an mACHR-6polypeptide or portion thereof such that treatment occurs. A nervoussystem disorder, smooth muscle related disorder, cardiac muscle relateddisorder or a gland related disorder can also be treated according tothe invention by administering to the subject having the disorder anucleic acid encoding an mACHR-6 polypeptide or portion thereof suchthat treatment occurs.

The invention also pertains to methods for detecting naturally occurringand recombinantly created genetic mutations in an mACHR-6 gene, therebydetermining if a subject with the mutated gene is at risk for (or ispredisposed to have) a disorder characterized by aberrant or abnormalmACHR-6 nucleic acid expression or mACHR-6 polypeptide activity, e.g., anervous system disorder, a smooth muscle related disorder, a cardiacmuscle related disorder or a gland related disorder. In preferredembodiments, the methods include detecting, in a sample of cells fromthe subject, the presence or absence of a genetic mutation characterizedby an alteration affecting the integrity of a gene encoding an mACHR-6polypeptide, or the misexpression of the mACHR-6 gene, such as thatcaused by a nucleic acid base substitution, deletion or addition, orgross sequence changes caused by a genetic translation, inversion orinsertion.

Another aspect of the invention pertains to methods for detecting thepresence of mACHR-6, or allelic variants thereof, in a biologicalsample. In a preferred embodiment, the methods involve contacting abiological sample (e.g., a brain or smooth muscle cell sample) with acompound or an agent capable of detecting mACHR-6 polypeptide or mACHR-6mRNA such that the presence of mACHR-6 is detected in the biologicalsample. The compound or agent can be, for example, a labeled orlabelable nucleic acid probe capable of hybridizing to mACHR-6 mRNA or alabeled or labelable antibody capable of binding to mACHR-6 polypeptide.The invention further provides methods for diagnosis of a subject with,for example, a nervous system disorder, a smooth muscle relateddisorder, a cardiac muscle related disorder or a gland related disorder,based on detection of mACHR-6 polypeptide or mRNA. In one embodiment,the method involves contacting a cell or tissue sample (e.g., a brain orsmooth muscle cell sample) from the subject with an agent capable ofdetecting mACHR-6 polypeptide or mRNA, determining the amount of mACHR-6polypeptide or mRNA expressed in the cell or tissue sample, comparingthe amount of mACHR-6 polypeptide or mRNA expressed in the cell ortissue sample to a control sample and forming a diagnosis based on theamount of mACHR-6 polypeptide or mRNA expressed in the cell or tissuesample as compared to the control sample. Preferably, the cell sample isa brain cell sample. Kits for detecting mACHR-6 in a biological samplewhich include agents capable of detecting mACHR-6 polypeptide or mRNAare also within the scope of the invention.

Still another aspect of the invention pertains to methods, e.g.,screening assays, for identifying a compound, e.g., a test compound, fortreating a disorder characterized by aberrant mACHR-6 nucleic acidexpression or polypeptide activity, e.g., a nervous system disorder, asmooth muscle related disorder, a cardiac muscle related disorder or agland related disorder. These methods typically include assaying theability of the compound or agent to modulate the expression of themACHR-6 gene or the activity of the mACHR-6 polypeptide therebyidentifying a compound for treating a disorder characterized by aberrantmACHR-6 nucleic acid expression or polypeptide activity. In a preferredembodiment, the method involves contacting a biological sample, e.g., acell or tissue sample, e.g., a brain or smooth muscle cell sample,obtained from a subject having the disorder with the compound or agent,determining the amount of mACHR-6 polypeptide expressed and/or measuringthe activity of the mACHR-6 polypeptide in the biological sample,comparing the amount of mACHR-6 polypeptide expressed in the biologicalsample and/or the measurable mACHR-6 biological activity in the cell tothat of a control sample. An alteration in the amount of mACHR-6polypeptide expression or mACHR-6 activity in the cell exposed to thecompound or agent in comparison to the control is indicative of amodulation of mACHR-6 expression and/or mACHR-6 activity.

The invention also pertains to methods for identifying a compound oragent, e.g., a test compound or agent, which interacts with (e.g., bindsto) an mACHR-6 polypeptide. These methods can include the steps ofcontacting the mACHR-6 polypeptide with the compound or agent underconditions which allow binding of the compound to the mACHR-6polypeptide to form a complex and detecting the formation of a complexof the mACHR-6 polypeptide and the compound in which the ability of thecompound to bind to the mACHR-6 polypeptide is indicated by the presenceof the compound in the complex.

The invention further pertains to methods for identifying a compound oragent, e.g., a test compound or agent, which modulates, e.g., stimulatesor inhibits, the interaction of the mACHR-6 polypeptide with a targetmolecule, e.g., acetylcholine, or a cellular protein involved inphosphatidylinositol turnover and signaling. In these methods, themACHR-6 polypeptide is contacted, in the presence of the compound oragent, with the target molecule under conditions which allow binding ofthe target molecule to the mACHR-6 polypeptide to form a complex. Analteration, e.g., an increase or decrease, in complex formation betweenthe mACHR-6 polypeptide and the target molecule as compared to theamount of complex formed in the absence of the compound or agent isindicative of the ability of the compound or agent to modulate theinteraction of the mACHR-6 polypeptide with a target molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the human mACHR-6 nucleotide (SEQ ID NO:1) and amino acid(SEQ ID NO:2) sequences. The coding region without the 5′ and 3′untranslated region of the human mACHR-6 gene is shown in SEQ ID NO:3.

FIG. 2 depicts the rat mACHR-6 nucleotide (SEQ ID NO:4) and amino acid(SEQ ID NO:5) sequences. The coding region without the 5′ and 3′untranslated region of the rat mACHR-6 gene is shown in SEQ ID NO:6.

FIG. 3 depicts the partial rat mACHR-6 nucleotide (SEQ ID NO:31) andamino acid (SEQ ID NO:32) sequences. The partial coding region withoutthe 3′ untranslated region of the rat mACHR-6 gene is shown in SEQ IDNO:33.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of novel molecules,referred to herein as mACHR-6 nucleic acid and polypeptide molecules,which play a role in or function in acetylcholine signaling pathways. Inone embodiment, the mACHR-6 molecules modulate the activity of one ormore proteins involved in a neurotransmitter signaling pathway, e.g., anacetylcholine signaling pathway. In a preferred embodiment, the mACHR-6molecules of the present invention are capable of modulating theactivity of proteins involved in the acetylcholine signaling pathway tothereby modulate the effects of acetylcholine on acetylcholineresponsive cells.

As used herein, the phrase “acetylcholine responsive cells” refers tocells which have a function which can be modulated (e.g., stimulated orinhibited) by the neurotransmitter acetylcholine. Examples of suchfunctions include mobilization of intracellular molecules whichparticipate in a signal transduction pathway, e.g., phosphatidylinositol4,5-bisphosphate (PIP₂) or inositol 1,4,5-triphosphate (IP₃),polarization of the plasma membrane, production or secretion ofmolecules, alteration in the structure of a cellular component, cellproliferation, cell migration, cell differentiation, and cell survival.Acetylcholine responsive cells preferably express an acetylcholinereceptor, e.g., a muscarinic receptor. Examples of acetylcholineresponsive cells include neural cells, e.g., central nervous system andperipheral nervous system cells (such as sympathetic and parasympatheticneurons); smooth muscle cells, e.g., smooth muscle cells in thedigestive tract, the urinary tract, the blood vessels, the airways andthe lungs, or the uterus; cardiac muscle cells, e.g., cardiomyocytes;and gland cells such as exocrine gland cells, e.g., pancreatic glandcells, e.g., pancreatic beta cells, tear gland cells, sweat gland cells,or parotid gland cells.

Depending on the type of cell, the response elicited by acetylcholine isdifferent. For example, in neural cells, acetylcholine regulates ionchannels, and neural signal to noise ratio. Inhibition or overstimulation of the activity of proteins involved in the acetylcholinesignaling pathway or misexpression of acetylcholine can lead to hypo- orhyperpolarization of the neural plasma membrane and to perturbed neuralsignal to noise ratio, which can in turn lead to nervous system relateddisorders. Examples of nervous system related disorders includecognitive disorders, e.g., memory and learning disorders, such asamnesia, apraxia, agnosia, amnestic dysnomia, amnestic spatialdisorientation, Kluver-Bucy syndrome, Alzheimer's related memory loss(Eglen R. M. (1996) Pharmacol. and Toxicol. 78(2):59-68; Perry E. K.(1995) Brain and Cognition 28(3):240-58) and learning disability;disorders affecting consciousness, e.g., visual hallucinations,perceptual disturbances, or delerium associated with Lewy body dementia;schitzo-effective disorders (Dean B. (1996) Mol. Psychiatry 1(l):54-8),schizophrenia with mood swings (Bymaster F. P. (1997) J. Clin.Psychiatry 58 (suppl.10):28-36; Yeomans J. S. (1995) Neuropharmacol.12(1):3-16; Reimann D. (1994) J. Psychiatric Res. 28(3):195-210),depressive illness (primary or secondary); affective disorders (JanowskyD. S. (1994) Am. J Med. Genetics 54(4):335-44); sleep disorders (KimuraF. (1997) J. Neurophysiol. 77(2):709-16), e.g., REM sleep abnormalitiesin patients suffering from, for example, depression (Riemann D. (1994)J. Psychosomatic Res. 38 Suppl. 1:15-25; Bourgin P. (1995) Neuroreport6(3): 532-6), paradoxical sleep abnormalities (Sakai K. (1997) Eur. JNeuroscience 9(3):415-23), sleep-wakefulness, and body temperature orrespiratory depression abnormalities during sleep (Shuman S. L. (1995)Am. J. Physiol. 269(2 Pt 2):R308-17; Mallick B. N. (1997) Eur. J. BrainRes. 750(1-2):311-7). Other examples of nervous system related disordersinclude disorders affecting pain generation mechanisms, e.g., painrelated to irritable bowel syndrome (Mitch C. H. (1997) J. Med Chem.40(4):538-46; Shannon H. E. (1997) J. Pharmac. and Exp. Therapeutics281(2):884-94; Bouaziz H. (1995) Anesthesia and Analgesia 80(6):1140-4;or Guimaraes A. P. (1994) Brain Res. 647(2):220-30) or chest pain;movement disorders (Monassi C. R. (1997) Physiol. and Behav.62(1):53-9), e.g., Parkinson's disease related movement disorders (FinnM. (1997) Pharmacol. Biochem. & Behavior 57(1-2):243-9; Mayorga A. J.(1997) Pharmacol. Biochem. & Behavior 56(2):273-9); eating disorders,e.g., insulin hypersecretion related obesity (Maccario M. (1997) J.Endocrinol. Invest. 20(1):8-12; Premawardhana L. D. (1994) Clin.Endocrinol. 40(5): 617-21); or drinking disorders, e.g., diabeticpolydipsia (Murzi E. (1997) Brain Res. 752(1-2):184-8; Yang X. (1994)Pharmacol. Biochem. & Behavior 49(1):1-6).

In smooth muscle, acetylcholine regulates (e.g., stimulates or inhibits)contraction. Inhibition or overstimulation of the activity of proteinsinvolved in the acetylcholine signaling pathway or misexpression ofacetylcholine can lead to smooth muscle related disorders such asirritable bowel syndrome, diverticular disease, urinary incontinence,oesophageal achalasia, or chronic obstructive airways disease.

In cardiac muscle, acetylcholine induces a reduction in the heart rateand in cardiac contractility. Inhibition or overstimulation of theactivity of proteins involved in the acetylcholine signaling pathway ormisexpression of acetylcholine can lead to heart muscle relateddisorders such as pathologic bradycardia or tachycardia, arrhythmia,flutter or fibrillation.

In glands such as exocrine glands, acetylcholine regulates the secretionof enzymes or hormones, e.g., in the parotid gland acetylcholine inducesthe release of amylase, and in the pancreas acetylcholine induces therelease of digestive enzymes and insulin. Inhibition or over stimulationof the activity of proteins involved in the acetylcholine signalingpathway or misexpression of acetylcholine can lead to gland relateddisorders such as xerostomia, or diabetes mellitus.

In a particularly preferred embodiment, the mACHR-6 molecules arecapable of modulating the activity of G proteins, as well asphosphatidylinositol metabolism and turnover in acetylcholine responsivecells. As used herein, a “G protein” is a protein which participates, asa secondary signal, in a variety of intracellular signal transductionpathways, e.g., in the acetylcholine signaling pathway primarily throughphosphatidylinositol metabolism and turnover. G proteins represent afamily of heterotrimeric proteins composed of α, β and γ subunits, whichbind guanine nucleotides. These proteins are usually linked to cellsurface receptors, e.g., receptors containing seven transmembranedomains, such as the muscarinic receptors. Following ligand binding tothe receptor, a conformational change is transmitted to the G protein,which causes the α-subunit to exchange a bound GDP molecule for a GTPmolecule and to dissociate from the βγ-subunits. The GTP-bound form ofthe a-subunit typically functions as an effector-modulating moiety,leading to the production of second messengers, such as cyclic AMP(e.g., by activation of adenylate cyclase), diacylglycerol or inositolphosphates. Greater than 20 different types of α-subunits are known inman, which associate with a smaller pool of β and γ subunits. Examplesof mammalian G proteins include Gi, Go, Gq, Gs and Gt. G proteins aredescribed extensively in Lodish H. et al. Molecular Cell Biology,(Scientific American Books Inc., New York, N.Y., 1995).

As used herein, “phosphatidylinositol turnover and metabolism” refers tothe molecules involved in the turnover and metabolism ofphosphatidylinositol 4,5-bisphosphate (PIP₂) as well as to theactivities of these molecules. PIP₂ is a phospholipid found in thecytosolic leaflet of the plasma membrane. Binding of acetylcholine to amuscarinic receptor activates the plasma-membrane enzyme phospholipase Cwhich in turn can hydrolyze PIP₂ to produce 1,2-diacylglycerol (DAG) andinositol 1,4,5-triphosphate (IP₃). Once formed IP₃ can diffuse to theendoplasmic reticulum surface where it can bind an IP₃ receptor, e.g., acalcium channel protein containing an IP₃ binding site. IP₃ binding caninduce opening of the channel, allowing calcium ions to be released intothe cytoplasm. IP₃ can also be phosphorylated by a specific kinase toform inositol 1,3,4,5-tetraphosphate (IP₄), a molecule which can causecalcium entry into the cytoplasm from the extracellular medium. IP₃ andIP₄ can subsequently be hydrolyzed very rapidly to the inactive productsinositol 1,4-biphosphate (IP₂) and inositol 1,3,4-triphosphate,respectively. These inactive products can be recycled by the cell tosynthesize PIP₂. The other second messenger produced by the hydrolysisof PIP₂, namely 1,2-diacylglycerol (DAG), remains in the cell membranewhere it can serve to activate the enzyme protein kinase C. Proteinkinase C is usually found soluble in the cytoplasm of the cell, but uponan increase in the intracellular calcium concentration, this enzyme canmove to the plasma membrane where it can be activated by DAG. Theactivation of protein kinase C in different cells results in variouscellular responses such as the phosphorylation of glycogen synthase, orthe phosphorylation of various transcription factors, e.g., NF-kB. Thelanguage “phosphatidylinositol activity”, as used herein, refers to anactivity of PIP₂ or one of its metabolites.

mACHR-6 nucleic acid molecules were identified by screening appropriatecDNA libraries (described in detail in Example 1). The rat mACHR-6nucleic acid molecule was identified by screening a rat brain cDNAlibrary. Positive clones were sequenced and the partial sequences wereanalyzed by comparison with sequences in a nucleic acid sequence database. This analysis indicated that the sequences were homologous to themuscarinic family of receptors. A longer rat clone was then isolated andsequenced. The human mACHR-6 nucleic acid molecule was identified byscreening a human cerebellum cDNA library using probes designed based onthe rat sequence.

Because of its ability to interact with (e.g., bind to) acetylcholine, Gproteins and other proteins involved in the acetylcholine signalingpathway, the mACHR-6 polypeptide is also a polypeptide which functionsin the acetylcholine signaling pathway.

The nucleotide sequence of the isolated human mACHR-6 cDNA and thepredicted amino acid sequence of the human mACHR-6 polypeptide are shownin FIG. 1 and in SEQ ID NOs:1 and 2, respectively. A plasmid containingthe full length nucleotide sequence encoding human mACHR-6 was depositedwith ATCC® on ______ and assigned Accession Number ______. This depositwill be maintained under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

The nucleotide sequence of the isolated rat mACHR-6 cDNA and thepredicted amino acid sequence of the rat mACHR-6 polypeptide are shownin FIG. 2 and in SEQ ID NOs:4 and 5, respectively. A plasmid containingthe full length nucleotide sequence encoding rat mACHR-6 was depositedwith ATCC® on ______ and assigned Accession Number ______. This depositwill be maintained under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

The nucleotide sequence of the isolated partial rat mACHR-6 cDNA and thepredicted amino acid sequence of the partial rat mACHR-6 polypeptide areshown in FIG. 3 and in SEQ ID NOs:31 and 32, respectively.

The human mACHR-6 gene, which is approximately 2689 nucleotides inlength, encodes a full length polypeptide having a molecular weight ofapproximately 51.2 KDa and which is approximately 445 amino acidresidues in length. The human mACHR-6 polypeptide is expressed at leastin the brain, in particular, regions of the brain such as thecerebellum, the cerebral cortex, the medulla, the occipital pole, thefrontal lobe, the temporal lobe, the putamen, the corpus callosum theamygdala, the caudate nucleus, the hippocampus, the substantia nigra,the subthalamic nucleus and the thalamus; spinal cord, placenta, lungs,spleen, liver, skeletal muscle, kidney, and testis. Based on structuralanalysis, amino acid residues 34-59 (SEQ ID NO:7), 73-91 (SEQ ID NO:8),109-130 (SEQ ID NO:9), 152-174 (SEQ ID NO:10), 197-219(SEQ ID NO:11),360-380 (SEQ ID NO:12), and 396-416 (SEQ ID NO:13) comprisetransmembrane domains. As used herein, the term “transmembrane domain”refers to a structural amino acid motif which includes a hydrophobichelix that spans the plasma membrane. A transmembrane domain alsopreferably includes a series of conserved serine, threonine, andtyrosine residues. For example, the transmembrane domains betweenresidues 109-130 (SEQ ID NO:9), 197-219 (SEQ ID NO:11), 360-380 (SEQ IDNO:12), and 396-416 (SEQ ID NO:13), contain threonine and tyrosineresidues (located about 1-2 helical turns away from the membranesurface), which are important for ligand, e.g., acetylcholine, binding.Other important residues in the transmembrane domains include theconserved aspartate residue in the transmembrane domain between residues109-130 (SEQ ID NO:9) and the conserved proline residue in thetransmembrane domain between residues 152-174 (SEQ ID NO:10), which arealso important for ligand, e.g., acetylcholine, binding. A skilledartisan will readily appreciate that the beginning and ending amino acidresidue recited for various domains/fragments of mACHR-6 are based onstructural analysis and that the actual beginning/ending amino acid foreach may vary by a few amino acids from that identified herein.

The rat mACHR-6 gene, which is approximately 3244 nucleotides in length,encodes a full length polypeptide having a molecular weight ofapproximately 51.2 kDa and which is at least about 445 amino acidresidues in length. The rat mACHR-6 polypeptide is expressed in thebrain. Amino acid residues 34-59 (SEQ ID NO:14), 73-91 (SEQ ID NO:15),109-130 (SEQ ID NO:16), 152-174 (SEQ ID NO:17), 197-219 (SEQ ID NO:18),360-380 (SEQ ID NO:19) and 396-416 (SEQ ID NO:20) comprise transmembranedomains.

The rat mACHR-6 gene, which is at least about 2218 nucleotides inlength, encodes a full length polypeptide having a molecular weight ofat least about 41.6 kDa and which is at least about 362 amino acidresidues in length. The rat mACHR-6 polypeptide is expressed in thebrain. Amino acid residues 1-8 (SEQ ID NO: 14), 26-47 (SEQ ID NO:15),69-91 (SEQ ID NO:16), 114-136 (SEQ ID NO:17), 277-297 (SEQ ID NO: 18),and 313-333 (SEQ ID NO: 19) comprise transmembrane domains.

The partial rat mACHR-6 gene, which is at least about 2218 nucleotidesin length, encodes a polypeptide having a molecular weight of at leastabout 41.6 kDa and which is at least about 362 amino acid residues inlength. The rat mACHR-6 polypeptide is expressed in the brain. Aminoacid residues 1-8 (SEQ ID NO:34), 26-47 (SEQ ID NO:35), 69-91 (SEQ IDNO:36), 114-136 (SEQ ID NO:37), 277-297 (SEQ ID NO:38), and 313-333 (SEQID NO:39) comprise transmembrane domains.

The mACHR-6 polypeptide, a biologically active portion or fragment ofthe polypeptide, or an allelic variant thereof can have one or more ofthe following mACHR-6 activities: 1) it can interact with (e.g., bindto) acetylcholine; 2) it can interact with (e.g., bind to) a G proteinor another protein which naturally binds to mACHR-6; 3) it can modulatethe activity of an ion channel (e.g., a potassium channel or a calciumchannel); 4) it can modulate cytosolic ion, e.g., calcium,concentration; 5) it can modulate the release of a neurotransmitter,e.g., acetylcholine, from a neuron, e.g., a presynaptic neuron; 6) itcan modulate an acetylcholine response in an acetylcholine responsivecell (e.g., a smooth muscle cell or a gland cell) to, for example,beneficially affect the acetylcholine responsive cell, e.g., a neuron;7) it can signal ligand binding via phosphatidylinositol turnover; and8) it can modulate, e.g., activate or inhibit, phospholipase C activity.

Various aspects of the invention are described in further detail in thefollowing subsections:

1. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode mACHR-6 or biologically active portions thereof, as well asnucleic acid fragments sufficient for use as hybridization probes toidentify mACHR-6-encoding nucleic acid (e.g., mACHR-6 mRNA). As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. An “isolated” nucleic acid moleculeis one which is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid. Preferably, an“isolated” nucleic acid is free of sequences which naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatedmACHR-6 nucleic acid molecule can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived (e.g., a hippocampal cell). Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized. A nucleic acid molecule of thepresent invention, e.g., a nucleic acid molecule having the nucleotidesequence of SEQ ID NO:1, 4, or 31, or a portion thereof, can be isolatedusing standard molecular biology techniques and the sequence informationprovided herein. For example, a human mACHR-6 cDNA can be isolated froma human hippocampus library using all or portion of SEQ ID NO:1, 4, or31 as a hybridization probe and standard hybridization techniques (e.g.,as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1, 4, or 31 can be isolated by the polymerase chain reaction usingoligonucleotide primers designed based upon the sequence of SEQ ID NO:1,4, or 31. For example, mRNA can be isolated from normal brain cells(e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwinet al. (1979) Biochemistry 18: 5294-5299) and cDNA can be prepared usingreverse transcriptase (e.g., Moloney MLV reverse transcriptase,available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase,available from Seikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for PCR amplification can be designed based uponthe nucleotide sequence shown in SEQ ID NO:1, 4, or 31. A nucleic acidof the invention can be amplified using cDNA or, alternatively, genomicDNA, as a template and appropriate oligonucleotide primers according tostandard PCR amplification techniques. The nucleic acid so amplified canbe cloned into an appropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to an mACHR-6nucleotide sequence can be prepared by standard synthetic techniques,e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:1, 4, or31 or the nucleotide sequence of the DNA insert of the plasmid depositedwith ATCC® as Accession Number ______. The sequence of SEQ ID NO:1corresponds to the human mACHR-6 cDNA. This cDNA comprises sequencesencoding the human mACHR-6 polypeptide (i.e., “the coding region”, fromnucleotides 291 to 1628 of SEQ ID NO:1), as well as 5′ untranslatedsequences (nucleotides 1 to 290 of SEQ ID NO:1) and 3′ untranslatedsequences (nucleotides 1629 to 2689 of SEQ ID NO:1). Alternatively, thenucleic acid molecule can comprise only the coding region of SEQ ID NO:1(e.g., nucleotides 291 to 1628 of SEQ ID NO:1, shown separately as SEQID NO:3). The sequence of SEQ ID NO:4 corresponds to the rat mACHR-6cDNA. This cDNA comprises sequences encoding the rat mACHR-6 polypeptide(i.e., “the coding region”, from nucleotides 778 to 2112 of SEQ IDNO:4), as well as 5′ untranslated sequences (nucleotides 1 to 777 of SEQID NO:4), and 3′ untranslated sequences (nucleotides 2113 to 3244 of SEQID NO:4). Alternatively, the nucleic acid molecule can comprise only thecoding region of SEQ ID NO:4 (e.g., nucleotides 778 to 2112 of SEQ IDNO:4, shown separately as SEQ ID NO:6). The sequence of SEQ ID NO:31corresponds to the partial rat mACHR-6 cDNA. This cDNA comprisessequences encoding part of the rat mACHR-6 polypeptide (i.e., part of“the coding region”, from nucleotides 1 to 1089 of SEQ ID NO:31), and 3′untranslated sequences (nucleotides 1090 to 2218 of SEQ ID NO:31).Alternatively, the nucleic acid molecule can comprise only the partialcoding region of SEQ ID NO:31 (e.g., nucleotides 1 to 1089, shownseparately as SEQ ID NO:33).

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:1, 4, or 31, the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC® asAccession Number ______, or a portion of either of these nucleotidesequences. A nucleic acid molecule which is complementary to thenucleotide sequence shown in SEQ ID NO:1, 4, or 31 is one which issufficiently complementary to the nucleotide sequence shown in SEQ IDNO:1, 4, or 31 such that it can hybridize to the nucleotide sequenceshown in SEQ ID NO:1, 4, or 31, respectively, thereby forming a stableduplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the invention comprises a nucleotide sequence which is at least about30-35%, preferably at least about 40-45%, more preferably at least about50-55%, even more preferably at least about 60-65%, yet more preferablyat least about 70-75%, still more preferably at least about 80-85%, andmost preferably at least about 90-95% or more homologous to thenucleotide sequence shown in SEQ ID NO:1, 4, or 31, or to the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC® asAccession Number ______, or a portion of these nucleotide sequences.Preferably, such nucleic acid molecules encode functionally active orinactive allelic variants of mACHR-6. In an additional preferredembodiment, an isolated nucleic acid molecule of the invention comprisesa nucleotide sequence which hybridizes, e.g., hybridizes under stringentconditions, to the nucleotide sequence shown in SEQ ID NO:1, 4, or 31,or the nucleotide sequence of the DNA insert of the plasmid depositedwith ATCC® as Accession Number ______, or a portion of either of thesenucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the coding region of SEQ ID NO:1, 4, or 31, for example afragment which can be used as a probe or primer or a fragment encoding abiologically active portion of mACHR-6. The nucleotide sequencedetermined from the cloning of the mACHR-6 gene from a mammal allows forthe generation of probes and primers designed for use in identifyingand/or cloning mACHR-6 homologues in other cell types, e.g., from othertissues, as well as mACHR-6 homologues from other mammals. Theprobe/primer typically comprises substantially purified oligonucleotide.The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12,preferably about 25, more preferably about 40, 50 or 75 consecutivenucleotides of SEQ ID NO: 1, 4, or 31 sense, an anti-sense sequence ofSEQ ID NO:1, 4, or 31, or naturally occurring mutants thereof. Primersbased on the nucleotide sequence in SEQ ID NO:1, 4, or 31 can be used inPCR reactions to clone mACHR-6 homologues. Probes based on the mACHR-6nucleotide sequences can be used to detect transcripts or genomicsequences encoding the same or homologous polypeptides. In preferredembodiments, the probe further comprises a label group attached thereto,e.g., the label group can be a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used as a part of adiagnostic test kit for identifying cells or tissue which misexpress anmACHR-6 polypeptide, such as by measuring a level of an mACHR-6-encodingnucleic acid in a sample of cells from a subject e.g., detecting mACHR-6mRNA levels or determining whether a genomic mACHR-6 gene has beenmutated or deleted.

In one embodiment, the nucleic acid molecule of the invention encodes apolypeptide or portion thereof which includes an amino acid sequencewhich is sufficiently homologous to an amino acid sequence of SEQ IDNO:2, 5, or 32 or an amino acid sequence encoded by the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC® asAccession Number ______ such that the polypeptide or portion thereofmaintains the ability to modulate an acetylcholine response in anacetylcholine responsive cell (e.g., naturally occurring allelicvariants of the rat and human mACHR-6 polypeptides described herein). Asused herein, the language “sufficiently homologous” refers topolypeptides or portions thereof which have amino acid sequences whichinclude a minimum number of identical or equivalent (e.g., an amino acidresidue which has a similar side chain as an amino acid residue in SEQID NO:2, 5, or 32) amino acid residues to an amino acid sequence of SEQID NO:2, 5, or 32 or an amino acid sequence encoded by the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC® asAccession Number ______ such that the polypeptide or portion thereof isable to modulate an acetylcholine response in an acetylcholineresponsive cell or a skilled artisan would clearly recognize it as anon-functional allelic variant of the rat and human mACHR-6 polypeptidesdescribed herein. Acetylcholine, as described herein, initiates avariety of responses in many different cell types. Examples of suchresponses are also described herein. In another embodiment, thepolypeptide is at least about 30-35%, preferably at least about 40-45%,more preferably at least about 50-55%, even more preferably at leastabout 60-65%, yet more preferably at least about 70-75%, still morepreferably at least about 80-85%, and most preferably at least about90-95% or more homologous to the amino acid sequence of SEQ ID NO:2, 5,or 32.

Portions of polypeptides encoded by the mACHR-6 nucleic acid molecule ofthe invention are preferably biologically active portions of the mACHR-6polypeptide. As used herein, the term “biologically active portion ofmACHR-6” is intended to include a portion, e.g., a domain/motif, ofmACHR-6 that has one or more of the following mACHR-6 activities: 1) itcan interact with (e.g., bind to) acetylcholine; 2) it can interact with(e.g., bind to) a G protein or another protein which naturally binds tomACHR-6; 3) it can modulate the activity of an ion channel (e.g., apotassium channel or a calcium channel); 4) it can modulate cytosolicion, e.g., calcium, concentration; 5) it can modulate the release of aneurotransmitter, e.g., acetylcholine, from a neuron, e.g., apresynaptic neuron; 6) it can modulate an acetylcholine response in anacetylcholine responsive cell (e.g., a smooth muscle cell or a glandcell) to, for example, beneficially affect the acetylcholine responsivecell, e.g., a neuron; 7) it can signal ligand binding viaphosphatidylinositol turnover; and 8) it can modulate, e.g., activate orinhibit, phospholipase C activity.

Standard binding assays, e.g., immunoprecipitations and yeast two-hybridassays as described herein, can be performed to determine the ability ofan mACHR-6 polypeptide or a biologically active portion thereof tointeract with (e.g., bind to) a binding partner such as a G protein. Todetermine whether an mACHR-6 polypeptide or a biologically activeportion thereof can modulate an acetylcholine response in anacetylcholine responsive cell, such cells can be transfected with aconstruct driving the overexpression of an mACHR-6 polypeptide or abiologically active portion thereof. Methods for the preparation ofacetylcholine responsive cells, e.g., intact smooth muscle cells orextracts from such cells are known in the art and described in Glukhovaet al. (1987) Tissue Cell 19 (5):657-63, Childs et al. (1992) J. Biol.Chem. 267 (32):22853-9, and White et al. (1996) J. Biol. Chem. 271(25):15008-17. The cells can be subsequently treated with acetylcholine,and a biological effect of acetylcholine on the cells, such asphosphatidylinositol turnover or cytosolic calcium concentration can bemeasured using methods known in the art (see Hartzell H. C. et al.(1988) Prog. Biophys. Mol. Biol. 52:165-247). Alternatively, transgenicanimals, e.g., mice overexpressing an mACHR-6 polypeptide or abiologically active portion thereof, can be used. Tissues from suchanimals can be obtained and treated with acetylcholine. For example,methods for preparing detergent-skinned muscle fiber bundles are knownin the art (Strauss et al. (1992) Am. J Physiol. 262:1437-45). Thecontractility of these tissues in response to acetylcholine can bedetermined using, for example, isometric force measurements as describedin Strauss et al., supra. Similarly, to determine whether an mACHR-6polypeptide or a biologically active portion thereof can modulate anacetylcholine response in an acetylcholine responsive cell such as agland cell, gland cells, e.g., parotid gland cells grown in tissueculture, can be transfected with a construct driving the overexpressionof an mACHR-6 polypeptide or a biologically active portion thereof. Thecells can be subsequently treated with acetylcholine, and the effect ofthe acetylcholine on amylase secretion from such cells can be determinedusing, for example an enzymatic assay with a labeled substrate. Thepreferred assays used for mACHR-6 activity will be based onphosphatidylinositol turnover such as those developed for the M1, M3 andM5 classes of receptors (see E. Watson et al. The G Protein LinkedReceptor: FactsBook (Academic Press, Boston, Mass., 1994), the contentsof which are incorporated herein by reference).

In one embodiment, the biologically active portion of mACHR-6 comprisesa transmembrane domain. Preferably, the transmembrane domain is encodedby a nucleic acid molecule derived from a human and is at least about50-55%, preferably at least about 60-65%, more preferably at least about70-75%, even more preferably at least about 80-85%, and most preferablyat least about 90-95% or more homologous to any of the transmembranedomains (i.e., amino acid residues 34-59, 109-130, 152-174, 197-219, or396-416) of SEQ ID NO:2 which are shown as separate sequences designatedSEQ ID NOs:7, 9, 10, 11, and 13, respectively, or to the rattransmembrane domains (i.e., amino acid residues 34-59, 73-91, 109-130,152-174, 197-219, 360-380, or 396-416 of SEQ ID NO:5 which are shown asseparate sequences designated SEQ ID NOs:14, 15,16, 17, 18, 19, and 20,respectively or amino acid residues 1-8, 26-47, 69-91, 114-136, 277-297,or 313-333 of SEQ ID NO:32 which are shown as separate sequencesdesignated SEQ ID NOs:34, 35, 36, 37, 38, or 39, respectively). Morepreferably, the transmembrane domain encoded by the human nucleic acidmolecule is at least about 75-80%, preferably at least about 80-85%,more preferably at least about 85-90%, and most preferably at leastabout 90-95% or more homologous to the transmembrane domain (i.e., aminoacid residues 360-380) of SEQ ID NO:2 which is shown as a separatesequence designated SEQ ID NO: 12, or at least about 80-85%, morepreferably at least about 85-90%, and most preferably at least about90-95% or more homologous to the transmembrane domain (i.e., amino acidresidues 73-91) of SEQ ID NO:2 which is shown as a separate sequencedesignated SEQ ID NO:8. In a preferred embodiment, the biologicallyactive portion of the polypeptide which includes the transmembranedomain can modulate the activity of a G protein or other binding partnerin a cell and/or modulate an acetylcholine response in an acetylcholineresponsive cell, e.g., a brain cell, to thereby beneficially affect thecell. In a preferred embodiment, the biologically active portioncomprises a transmembrane domain of the human mACHR-6 as represented byamino acid residues 34-59 (SEQ ID NO:7), 73-91 (SEQ ID NO:8), 109-130(SEQ ID NO:9), 152-174 (SEQ ID NO:10), 197-219 (SEQ ID NO:11), 360-380(SEQ ID NO:12), and 396-416 (SEQ ID NO:13), a transmembrane domain ofthe full length rat mACHR-6 as represented by amino acid residues 34-59(SEQ ID NO: 14), 73-91 (SEQ ID NO: 15), 109-130 (SEQ ID NO: 16), 152-174(SEQ ID NO: 17), 197-219 (SEQ ID NO: 18), 360-380 (SEQ ID NO: 19), and396-416 (SEQ ID NO:20), or a transmembrane domain of the partial ratmACHR-6 as represented by amino residues 1-8 (SEQ ID NO:34), 26-47 (SEQID NO:35), 69-91 (SEQ ID NO:36), 114-136 (SEQ ID NO:37), 277-297 (SEQ IDNO:38), and 313-333 (SEQ ID NO:39). Additional nucleic acid fragmentsencoding biologically active portions of mACHR-6 can be prepared byisolating a portion of SEQ ID NO:1, 4, or 31, expressing the encodedportion of mACHR-6 polypeptide or peptide (e.g., by recombinantexpression in vitro) and assessing the activity of the encoded portionof mACHR-6 polypeptide or peptide.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1, 4, or 31 (andportions thereof) due to degeneracy of the genetic code and thus encodethe same mACHR-6 polypeptide as that encoded by the nucleotide sequenceshown in SEQ ID NO:1, 4, or 31. In another embodiment, an isolatednucleic acid molecule of the invention has a nucleotide sequenceencoding a polypeptide having an amino acid sequence shown in SEQ IDNO:2, 5, or 32 or a polypeptide having an amino acid sequence encoded bythe nucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______. In a still further embodiment, thenucleic acid molecule of the invention encodes a full length humanpolypeptide which is substantially homologous to the amino acid sequenceof SEQ ID NO:2 or 4 (encoded by the open reading frame shown in SEQ IDNO:3, 6, or 33, respectively) or an amino acid sequence encoded by thenucleotide sequence of the DNA insert of the plasmid deposited withATCC® as Accession Number ______.

In addition to the mACHR-6 nucleotide sequence shown in SEQ ID NO:1, 4,or 31, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof mACHR-6 may exist within a population (e.g., the human population).Such genetic polymorphism in the mACHR-6 gene may exist amongindividuals within a population due to natural allelic variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding an mACHR-6polypeptide, preferably a mammalian mACHR-6 polypeptide. Such naturalallelic variations can typically result in 1-5% variance in thenucleotide sequence of the mACHR-6 gene. Any and all such nucleotidevariations and resulting amino acid polymorphisms in mACHR-6 that arethe result of natural allelic variation are intended to be within thescope of the invention. Such allelic variation includes both activeallelic variants as well as non-active or reduced activity allelicvariants, the later two types typically giving rise to a pathologicaldisorder. Moreover, nucleic acid molecules encoding mACHR-6 polypeptidesfrom other species, and thus which have a nucleotide sequence whichdiffers from the human sequence of SEQ ID NO:1, are intended to bewithin the scope of the invention. Nucleic acid molecules correspondingto natural allelic variants and non-human homologues of the humanmACHR-6 cDNA of the invention can be isolated based on their homology tothe human mACHR-6 nucleic acid disclosed herein using the human cDNA, ora portion thereof, as a hybridization probe according to standardhybridization techniques under stringent hybridization conditions.Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15 nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1 or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC® as Accession Number ______.In other embodiments, the nucleic acid is at least 30, 50, 100, 250,300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length. Asused herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% homologous to each othertypically remain hybridized to each other. Preferably, the conditionsare such that sequences at least about 65%, more preferably at leastabout 70%, and even more preferably at least about 75% or morehomologous to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringenthybridization conditions are hybridization in 6×sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions tothe sequence of SEQ ID NO:1 corresponds to a naturally-occurring nucleicacid molecule. As used herein, a “naturally-occurring” nucleic acidmolecule refers to an RNA or DNA molecule having a nucleotide sequencethat occurs in nature (e.g., encodes a natural polypeptide). In oneembodiment, the nucleic acid encodes a natural human mACHR-6.

In addition to naturally-occurring allelic variants of the mACHR-6sequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequence of SEQ ID NO:1, 4, or 31, thereby leading to changesin the amino acid sequence of the encoded mACHR-6 polypeptide, withoutaltering the functional ability of the mACHR-6 polypeptide. For example,nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of SEQID NO:1, 4, or 31. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence of mACHR-6 (e.g., thesequence of SEQ ID NO:2, 5, or 32) without altering the activity ofmACHR-6, whereas an “essential” amino acid residue is required formACHR-6 activity. For example, conserved amino acid residues, e.g.,aspartates, prolines threonines and tyrosines, in the transmembranedomains of mACHR-6 are most likely important for binding toacetylcholine and are thus essential residues of mACHR-6. Other aminoacid residues, however, (e.g., those that are not conserved or onlysemi-conserved in the transmembrane domain) may not be essential foractivity and thus are likely to be amenable to alteration withoutaltering mACHR-6 activity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding mACHR-6 polypeptides that contain changes in aminoacid residues that are not essential for mACHR-6 activity. Such mACHR-6polypeptides differ in amino acid sequence from SEQ ID NO:2, 5, or 32yet retain at least one of the mACHR-6 activities described herein. Inone embodiment, the isolated nucleic acid molecule comprises anucleotide sequence encoding a polypeptide, wherein the polypeptidecomprises an amino acid sequence at least about 30-35%, preferably atleast about 40-45%, more preferably at least about 50-55%, even morepreferably at least about 60-65%, yet more preferably at least about70-75%, still more preferably at least about 80-85%, and most preferablyat least about 90-95% or more homologous to the amino acid sequence ofSEQ ID NO:2, 5, or 32.

To determine the percent homology of two amino acid sequences (e.g., SEQID NO:2, 5, or 32 and a mutant form thereof) or of two nucleic acids,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in the sequence of one polypeptide or nucleic acid foroptimal alignment with the other polypeptide or nucleic acid). The aminoacid residues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in one sequence(e.g., SEQ ID NO:2, 5, or 32) is occupied by the same amino acid residueor nucleotide as the corresponding position in the other sequence (e.g.,a mutant form of mACHR-6), then the molecules are homologous at thatposition (i.e., as used herein amino acid or nucleic acid “homology” isequivalent to amino acid or nucleic acid “identity”). The percenthomology between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100).

The determination of percent homology between two sequences can beaccomplished using a mathematical algorithim. A preferred, non-limitingexample of a mathematical algorithim utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol.Biol. 215:403-10. BLAST nucleotide searches can be performed performedwith the NBLAST program, score=100, wordlength=12 to obtain nucleotidesequences homologous to mACHR-6 nucleic acid molecules of the invention.BLAST protein searches can be performed with the XBLAST program,score=50, wordlength=3 to obtain amino acid sequences homologous tomACHR-6 protein molecules of the invention. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST can be utilized as described inAltschul et al., (1997) Nucleic Acids Research 25(17):3389-3402. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nim.nih.gov. Another preferred, non-limiting example ofa mathematical algorithim utilized for the comparison of sequences isthe algorithm of Myers and Miller, CABIOS (1989). Such an algorithm isincorporated into the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

An isolated nucleic acid molecule encoding an mACHR-6 polypeptidehomologous to the polypeptide of SEQ ID NO:2, 5, or 32 can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of SEQ ID NO:1, 4, or 31, respectively,such that one or more amino acid substitutions, additions or deletionsare introduced into the encoded polypeptide. Mutations can be introducedinto SEQ ID NO:1, 4, or 31 by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. Preferably, conservative aminoacid substitutions are made at one or more predicted non-essential aminoacid residues. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), non-polar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in mACHR-6is preferably replaced with another amino acid residue from the sameside chain family. Alternatively, in another embodiment, mutations canbe introduced randomly along all or part of an mACHR-6 coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for an mACHR-6 activity described herein to identify mutantsthat retain mACHR-6 activity. Following mutagenesis of SEQ ID NO:1, 4,or 31, the encoded polypeptide can be expressed recombinantly (e.g., asdescribed in Examples 3 and 4) and the activity of the polypeptide canbe determined using, for example, assays described herein.

In addition to the nucleic acid molecules encoding mACHR-6 polypeptidesdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a polypeptide, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire mACHR-6 coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding mACHR-6.

The term “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues, e.g.,the entire coding region of SEQ ID NO:1 comprises nucleotides 291 to1628 (shown separately as SEQ ID NO:3) and the coding region of SEQ IDNO:4 comprises nucleotides 778 to 2112 (shown separately as SEQ IDNO:6). In another embodiment, the antisense nucleic acid molecule isantisense to a “noncoding region” of the coding strand of a nucleotidesequence encoding mACHR-6. The term “noncoding region” refers to 5′ and3′ sequences which flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding mACHR-6 disclosed herein(e.g., SEQ ID NOs:1, 4, and 31), antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crickbase pairing. The antisense nucleic acid molecule can be complementaryto the entire coding region of mACHR-6 mRNA, but more preferably is anoligonucleotide which is antisense to only a portion of the coding ornoncoding region of mACHR-6 mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of mACHR-6 mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding an mACHR-6polypeptide to thereby inhibit expression of the polypeptide, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of anantisense nucleic acid molecule of the invention includes directinjection at a tissue site. Alternatively, an antisense nucleic acidmolecule can be modified to target selected cells and then administeredsystemically. For example, for systemic administration, an antisensemolecule can be modified such that it specifically binds to a receptoror an antigen expressed on a selected cell surface, e.g., by linking theantisense nucleic acid molecule to a peptide or an antibody which bindsto a cell surface receptor or antigen. The antisense nucleic acidmolecule can also be delivered to cells using the vectors describedherein. To achieve sufficient intracellular concentrations of theantisense molecules, vector constructs in which the antisense nucleicacid molecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleavemACHR-6 mRNA transcripts to thereby inhibit translation of mACHR-6 mRNA.A ribozyme having specificity for an mACHR-6-encoding nucleic acid canbe designed based upon the nucleotide sequence of an mACHR-6 cDNAdisclosed herein (i.e., SEQ ID NO: 1, 4, or 31). For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in an mACHR-6-encoding mRNA. See,e.g., Cech et al. U.S. Pat. No. 4,987,071 and Cech et al. U.S. Pat. No.5,116,742. Alternatively, mACHR-6 mRNA can be used to select a catalyticRNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science261:1411-1418.

Alternatively, mACHR-6 gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of themACHR-6 (e.g., the mACHR-6 promoter and/or enhancers) to form triplehelical structures that prevent transcription of the mACHR-6 gene intarget cells. See generally, Helene, C. (1991) Anticancer Drug Des.6(6):569-84; Helene, C. et al. (1992) Ann. N.Y Acad. Sci. 660:27-36; andMaher, L. J. (1992) Bioassays 14(12):807-15.

II. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding mACHR-6 (or aportion thereof). As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology. Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of polypeptide desired, etc. The expression vectorsof the invention can be introduced into host cells to thereby producepolypeptides or peptides, including fusion polypeptides or peptides,encoded by nucleic acids as described herein (e.g., mACHR-6polypeptides, mutant forms of mACHR-6, fusion polypeptides, and thelike).

The recombinant expression vectors of the invention can be designed forexpression of mACHR-6 in prokaryotic or eukaryotic cells. For example,mACHR-6 can be expressed in bacterial cells such as E. coli, insectcells (e.g., using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology. Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of polypeptides in prokaryotes is most often carried out inE. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion polypeptides.Fusion vectors add a number of amino acids to a polypeptide encodedtherein, usually to the amino terminus of the recombinant polypeptide.Such fusion vectors typically serve three purposes: 1) to increaseexpression of recombinant polypeptide; 2) to increase the solubility ofthe recombinant polypeptide; and 3) to aid in the purification of therecombinant polypeptide by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantpolypeptide to enable separation of the recombinant polypeptide from thefusion moiety subsequent to purification of the fusion polypeptide. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to thetarget recombinant polypeptide. In one embodiment, the coding sequenceof the mACHR-6 is cloned into a pGEX expression vector to create avector encoding a fusion polypeptide comprising, from the N-terminus tothe C-terminus, GST-thrombin cleavage site-mACHR-6. The fusionpolypeptide can be purified by affinity chromatography usingglutathione-agarose resin. Recombinant mACHR-6 unfused to GST can berecovered by cleavage of the fusion polypeptide with thrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn 10-lac fusion promoter mediated bya coexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant polypeptide expression in E. coliis to express the polypeptide in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant polypeptide(Gottesman, S., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 119-128). Another strategy isto alter the nucleic acid sequence of the nucleic acid to be insertedinto an expression vector so that the individual codons for each aminoacid are those preferentially utilized in E. coli (Wada et al. (1992)Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acidsequences of the invention can be carried out by standard DNA synthesistechniques.

In another embodiment, the mACHR-6 expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari, et al., (1987) Embo J 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

Alternatively, mACHR-6 can be expressed in insect cells using, forexample, baculovirus expression vectors. Baculovirus vectors availablefor expression of polypeptides in cultured insect cells (e.g., Sf 9cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to mACHR-6 mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example,mACHR-6 polypeptide can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning. A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G4 18, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding mACHR-6 or can be introduced on a separatevector. Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) mACHR-6polypeptide. Accordingly, the invention further provides methods forproducing mACHR-6 polypeptide using the host cells of the invention. Inone embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding mACHR-6has been introduced) in a suitable medium until mACHR-6 is produced. Inanother embodiment, the method further comprises isolating mACHR-6 fromthe medium or the host cell.

The host cells of the invention can also be used to produce non-humantransgenic animals. The non-human transgenic animals can be used inscreening assays designed to identify agents or compounds, e.g., drugs,pharmaceuticals, etc., which are capable of ameliorating detrimentalsymptoms of selected disorders such as nervous system disorders, smoothmuscle related disorders, cardiac muscle related disorders and glandrelated disorders. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichmACHR-6-coding sequences have been introduced. Such host cells can thenbe used to create non-human transgenic animals in which exogenousmACHR-6 sequences have been introduced into their genome or homologousrecombinant animals in which endogenous mACHR-6 sequences have beenaltered. Such animals are useful for studying the function and/oractivity of mACHR-6 and for identifying and/or evaluating modulators ofmACHR-6 activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal include atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, and the like.A transgene is exogenous DNA which is integrated into the genome of acell from which a transgenic animal develops and which remains in thegenome of the mature animal, thereby directing the expression of anencoded gene product in one or more cell types or tissues of thetransgenic animal. As used herein, a “homologous recombinant animal” isa non-human animal, preferably a mammal, more preferably a mouse, inwhich an endogenous mACHR-6 gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

A transgenic animal of the invention can be created by introducingmACHR-6-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The humanmACHR-6 cDNA sequence of SEQ ID NO:1 can be introduced as a transgeneinto the genome of a non-human animal. Furthermore, the rat mACHR-6 cDNAsequence of SEQ ID NO:4 can be introduced as a transgene into the genomeof a non-rat animal. Moreover, a non-human homologue of the humanmACHR-6 gene, such as a mouse mACHR-6 gene, can be isolated based onhybridization to the human or rat mACHR-6 cDNA (described further insubsection I above) and used as a transgene. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to themACHR-6 transgene to direct expression of mACHR-6 polypeptide toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the mACHR-6 transgene in its genome and/or expression ofmACHR-6 mRNA in tissues or cells of the animals. A transgenic founderanimal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encodingmACHR-6 can further be bred to other transgenic animals carrying othertransgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of an mACHR-6 gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the mACHR-6 gene. The mACHR-6 gene can be a humangene (e.g., from a human genomic clone isolated from a human genomiclibrary screened with the cDNA of SEQ ID NO:1), but more preferably, isa rat mACHR-6 gene of SEQ ID NO:4 or 31, or another non-human homologueof a human mACHR-6 gene. For example, a mouse mACHR-6 gene can beisolated from a mouse genomic DNA library using the mACHR-6 cDNA of SEQID NO:1, 4, or 31 as a probe. The mouse mACHR-6 gene then can be used toconstruct a homologous recombination vector suitable for altering anendogenous mACHR-6 gene in the mouse genome. In a preferred embodiment,the vector is designed such that, upon homologous recombination, theendogenous mACHR-6 gene is functionally disrupted (i.e., no longerencodes a functional polypeptide; also referred to as a “knock out”vector). Alternatively, the vector can be designed such that, uponhomologous recombination, the endogenous mACHR-6 gene is mutated orotherwise altered but still encodes functional polypeptide (e.g., theupstream regulatory region can be altered to thereby alter theexpression of the endogenous mACHR-6 polypeptide). In the homologousrecombination vector, the altered portion of the mACHR-6 gene is flankedat its 5′ and 3′ ends by additional nucleic acid of the mACHR-6 gene toallow for homologous recombination to occur between the exogenousmACHR-6 gene carried by the vector and an endogenous mACHR-6 gene in anembryonic stem cell. The additional flanking mACHR-6 nucleic acid is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector (see for example, Thomas,K. R. and Capecchi, M. R. (1987) Cell 51:503 for a description ofhomologous recombination vectors). The vector is introduced into anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced mACHR-6 gene has homologously recombined with theendogenous mACHR-6 gene are selected (see e.g., Li, E. et al. (1992)Cell 69:915). The selected cells are then injected into a blastocyst ofan animal (e.g., a mouse) to form aggregation chimeras (see e.g.,Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission-of the transgene. Methods forconstructing homologous recombination vectors and homologous recombinantanimals are described further in Bradley, A. (1991) Current Opinion inBiotechnology 2:823-829 and in PCT International Publication Nos. WO90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) PNAS 89:6232-6236.Another example of a recombinase system is the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355.If a cre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected polypeptide are required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected polypeptide and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g., the somatic cell, isisolated.

III. Isolated mACHR-6 Polypeptides and Anti-mACHR-6 Antibodies

Another aspect of the invention pertains to isolated mACHR-6polypeptides, and biologically active portions thereof, as well aspeptide fragments suitable for use as immunogens to raise anti-mACHR-6antibodies. An “isolated” or “purified” polypeptide or biologicallyactive portion thereof is substantially free of cellular material whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. The language “substantially freeof cellular material” includes preparations of mACHR-6 polypeptide inwhich the polypeptide is separated from cellular components of the cellsin which it is naturally or recombinantly produced. In one embodiment,the language “substantially free of cellular material” includespreparations of mACHR-6 polypeptide having less than about 30% (by dryweight) of non-mACHR-6 polypeptide (also referred to herein as a“contaminating polypeptide”), more preferably less than about 20% ofnon-mACHR-6 polypeptide, still more preferably less than about 10% ofnon-mACHR-6 polypeptide, and most preferably less than about 5%non-mACHR-6 polypeptide. When the mACHR-6 polypeptide or biologicallyactive portion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the polypeptidepreparation. The language “substantially free of chemical precursors orother chemicals” includes preparations of mACHR-6 polypeptide in whichthe polypeptide is separated from chemical precursors or other chemicalswhich are involved in the synthesis of the polypeptide. In oneembodiment, the language “substantially free of chemical precursors orother chemicals” includes preparations of mACHR-6 polypeptide havingless than about 30% (by dry weight) of chemical precursors ornon-mACHR-6 chemicals, more preferably less than about 20% chemicalprecursors or non-mACHR-6 chemicals, still more preferably less thanabout 10% chemical precursors or non-mACHR-6 chemicals, and mostpreferably less than about 5% chemical precursors or non-mACHR-6chemicals. In preferred embodiments, isolated polypeptides orbiologically active portions thereof lack contaminating polypeptidesfrom the same animal from which the mACHR-6 polypeptide is derived.Typically, such polypeptides are produced by recombinant expression of,for example, a human mACHR-6 polypeptide in a non-human cell.

An isolated mACHR-6 polypeptide or a portion thereof of the inventioncan modulate an acetylcholine response in an acetylcholine responsivecell or be a naturally occurring, non-functional allelic variant of anmACHR-6 polypeptide. In preferred embodiments, the polypeptide orportion thereof comprises an amino acid sequence which is sufficientlyhomologous to an amino acid sequence of SEQ ID NO:2, 5, or 32 such thatthe polypeptide or portion thereof maintains the ability to modulate anacetylcholine response in an acetylcholine responsive cell. The portionof the polypeptide is preferably a biologically active portion asdescribed herein. In another preferred embodiment, the human mACHR-6polypeptide (i.e., amino acid residues 1-398 of SEQ ID NO:2) or the ratmACHR-6 polypeptide (i.e., amino acid residues 1-445 of SEQ ID NO:5 oramino acid residues 1-401 of SEQ ID NO:32) has an amino acid sequenceshown in SEQ ID NO:2, 5, or 32, respectively, or an amino acid sequencewhich is encoded by the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC® as Accession Number ______. In yet anotherpreferred embodiment, the mACHR-6 polypeptide has an amino acid sequencewhich is encoded by a nucleotide sequence which hybridizes, e.g.,hybridizes under stringent conditions, to the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC® as Accession Number______. In still another preferred embodiment, the mACHR-6 polypeptidehas an amino acid sequence which is encoded by a nucleotide sequencethat is at least about 30-35%, preferably at least about 40-45%, morepreferably at least about 50-55%, even more preferably at least about60-65%, yet more preferably at least about 70-75%, still more preferablyat least about 80-85%, and most preferably at least about 90-95% or morehomologous to the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC® as Accession Number ______. The preferred mACHR-6polypeptides of the present invention also preferably possess at leastone of the mACHR-6 activities described herein. For example, a preferredmACHR-6 polypeptide of the present invention includes an amino acidsequence encoded by a nucleotide sequence which hybridizes, e.g.,hybridizes under stringent conditions, to the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC® as Accession Number______ and which can modulate an acetylcholine response in anacetylcholine responsive cell.

In other embodiments, the mACHR-6 polypeptide is substantiallyhomologous to the amino acid sequence of SEQ ID NO:2, 5, or 32 andretains the functional activity of the polypeptide of SEQ ID NO:2, 5, or32 yet differs in amino acid sequence due to natural allelic variationor mutagenesis, as described in detail in subsection I above.Accordingly, in another embodiment, the mACHR-6 polypeptide is apolypeptide which comprises an amino acid sequence which is at leastabout 30-35%, preferably at least about 40-45%, more preferably at leastabout 50-55%, even more preferably at least about 60-65%, yet morepreferably at least about 70-75%, still more preferably at least about80-85%, and most preferably at least about 90-95% or more homologous tothe amino acid sequence of SEQ ID NO:2, 5, or 32 and which has at leastone of the mACHR-6 activities described herein. In still otherembodiments, the invention pertains to a full length human polypeptidewhich is substantially homologous to the entire amino acid sequence ofSEQ ID NO:2, 5, or 32. In still another embodiment, the inventionpertains to nonfunctional, naturally occurring allelic variants of themACHR-6 polypeptides described herein. Such allelic variants willtypically contain a non-conservative substitution, a deletion, orinsertion or premature truncation of the amino acid sequence of SEQ IDNO:2, 5, or 32.

Biologically active portions of the mACHR-6 polypeptide include peptidescomprising amino acid sequences derived from the amino acid sequence ofthe mACHR-6 polypeptide, e.g., the amino acid sequence shown in SEQ IDNO:2, 5, or 32 or the amino acid sequence of a polypeptide homologous tothe mACHR-6 polypeptide, which include less amino acids than the fulllength mACHR-6 polypeptide or the full length polypeptide which ishomologous to the mACHR-6 polypeptide, and exhibit at least one activityof the mACHR-6 polypeptide. Typically, biologically active portions(peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35,36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise adomain or motif, e.g., a transmembrane domain, with at least oneactivity of the mACHR-6 polypeptide. Preferably, the domain is atransmembrane domain derived from a human and is at least about 75-80%,preferably at least about 80-85%, more preferably at least about 85-90%,and most preferably at least about 90-95% or more homologous to SEQ IDNO:7, 8, 9, 10, 11, 12, or 13 or to the corresponding rat sequences. Ina preferred embodiment, the biologically active portion of thepolypeptide which includes the transmembrane domain can modulate theactivity of a G protein in a cell and/or modulate an acetylcholineresponse in a cell, e.g., an acetylcholine responsive cell, e.g., abrain cell, to thereby beneficially affect the acetylcholine responsivecell. In a preferred embodiment, the biologically active portioncomprises a transmembrane domain of mACHR-6 as represented by amino acidresidues 34-59 (SEQ ID NO:7), 73-91 (SEQ ID NO:8), 109-130 (SEQ IDNO:9), 152-174 (SEQ ID NO:10), 197-219 (SEQ ID NO:11), 360-380 (SEQ IDNO:12), and 396-416 (SEQ ID NO: 13), or the corresponding rat sequencesshown in SEQ ID NOs:14-20 and 34-39. Moreover, other biologically activeportions, in which other regions of the polypeptide are deleted, can beprepared by recombinant techniques and evaluated for one or more of theactivities described herein. Preferably, the biologically activeportions of the mACHR-6 polypeptide include one or more selecteddomains/motifs or portions thereof having biological activity.

mACHR-6 polypeptides are preferably produced by recombinant DNAtechniques. For example, a nucleic acid molecule encoding thepolypeptide is cloned into an expression vector (as described above),the expression vector is introduced into a host cell (as describedabove) and the mACHR-6 polypeptide is expressed in the host cell. ThemACHR-6 polypeptide can then be isolated from the cells by anappropriate purification scheme using standard polypeptide purificationtechniques. Alternative to recombinant expression, an mACHR-6polypeptide, protein, or peptide can be synthesized chemically usingstandard peptide synthesis techniques. Moreover, native mACHR-6polypeptide can be isolated from cells (e.g., hippocampal cells,substantia nigra cells, or parotid gland cells), for example using ananti-mACHR-6 antibody (described further below).

The invention also provides mACHR-6 chimeric or fusion polypeptides. Asused herein, an mACHR-6 “chimeric polypeptide” or “fusion polypeptide”comprises an mACHR-6 polypeptide operatively linked to a non-mACHR-6polypeptide. An “mACHR-6 polypeptide” refers to a polypeptide having anamino acid sequence corresponding to mACHR-6, whereas a “non-mACHR-6polypeptide” refers to a heterologous polypeptide having an amino acidsequence corresponding to a polypeptide which is not substantiallyhomologous to the mACHR-6 polypeptide, e.g., a polypeptide which isdifferent from the mACHR-6 polypeptide and which is derived from thesame or a different organism. Within the fusion polypeptide, the term“operatively linked” is intended to indicate that the mACHR-6polypeptide and the non-mACHR-6 polypeptide are fused in-frame to eachother. The non-mACHR-6 polypeptide can be fused to the N-terminus orC-terminus of the mACHR-6 polypeptide. For example, in one embodimentthe fusion polypeptide is a GST-mACHR-6 fusion polypeptide in which themACHR-6 sequences are fused to the C-terminus of the GST sequences.Other types of fusion polypeptides include, but are not limited to,enzymatic fusion polypeptides, for example beta-galactosidase fusions,yeast two-hybrid GAL fusions, poly His fusions and Ig fusions. Suchfusion polypeptides, particularly poly His fusions, can facilitate thepurification of recombinant mACHR-6. In another embodiment, the fusionpolypeptide is an mACHR-6 polypeptide containing a heterologous signalsequence at its N-terminus. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of mACHR-6 can be increased throughuse of a heterologous signal sequence.

Preferably, an mACHR-6 chimeric or fusion polypeptide of the inventionis produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and re-amplified togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). AnmACHR-6-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the mACHR-6polypeptide.

The present invention also pertains to homologues of the mACHR-6polypeptides which function as either an mACHR-6 agonist (mimetic) or anmACHR-6 antagonist. In a preferred embodiment, the mACHR-6 agonists andantagonists stimulate or inhibit, respectively, a subset of thebiological activities of the naturally occurring form of the mACHR-6polypeptide. Thus, specific biological effects can be elicited bytreatment with a homologue of limited function. In one embodiment,treatment of a subject with a homologue having a subset of thebiological activities of the naturally occurring form of the polypeptidehas fewer side effects in a subject relative to treatment with thenaturally occurring form of the mACHR-6 polypeptide.

Homologues of the mACHR-6 polypeptide can be generated by mutagenesis,e.g., discrete point mutation or truncation of the mACHR-6 polypeptide.As used herein, the term “homologue” refers to a variant form of themACHR-6 polypeptide which acts as an agonist or antagonist of theactivity of the mACHR-6 polypeptide. An agonist of the mACHR-6polypeptide can retain substantially the same, or a subset, of thebiological activities of the mACHR-6 polypeptide. An antagonist of themACHR-6 polypeptide can inhibit one or more of the activities of thenaturally occurring form of the mACHR-6 polypeptide, by, for example,competitively binding to a downstream or upstream member of the mACHR-6cascade which includes the mACHR-6 polypeptide. Thus, the mammalianmACHR-6 polypeptide and homologues thereof of the present invention canbe either positive or negative regulators of acetylcholine responses inacetylcholine responsive cells.

In an alternative embodiment, homologues of the mACHR-6 polypeptide canbe identified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the mACHR-6 polypeptide for mACHR-6 polypeptideagonist or antagonist activity. In one embodiment, a variegated libraryof mACHR-6 variants is generated by combinatorial mutagenesis at thenucleic acid level and is encoded by a variegated gene library. Avariegated library of mACHR-6 variants can be produced by, for example,enzymatically ligating a mixture of synthetic oligonucleotides into genesequences such that a degenerate set of potential mACHR-6 sequences isexpressible as individual polypeptides, or alternatively, as a set oflarger fusion polypeptides (e.g., for phage display) containing the setof mACHR-6 sequences therein. There are a variety of methods which canbe used to produce libraries of potential mACHR-6 homologues from adegenerate oligonucleotide sequence. Chemical synthesis of a degenerategene sequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential mACHR-6sequences. Methods for synthesizing degenerate oligonucleotides areknown in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3;Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakuraetal. (1984)Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the mACHR-6 polypeptide codingcan be used to generate a variegated population of mACHR-6 fragments forscreening and subsequent selection of homologues of an mACHR-6polypeptide. In one embodiment, a library of coding sequence fragmentscan be generated by treating a double stranded PCR fragment of anmACHR-6 coding sequence with a nuclease under conditions wherein nickingoccurs only about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with SI nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal, C-terminal and internal fragments of various sizes of themACHR-6 polypeptide.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of mACHR-6 homologues. Themost widely used techniques, which are amenable to high through-putanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recrusive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify mACHR-6 homologues (Arkin and Yourvan (1992) PNAS 89:7811-7815;Delgrave et al. (1993) Protein Engineering 6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated mACHR-6 library.

For example, a library of expression vectors can be transfected into acell line ordinarily responsive to acetylcholine. The transfected cellsare then contacted with acetylcholine and the effect of the mACHR-6mutant on signaling by acetylcholine can be detected, e.g., by measuringintracellular calcium concentration. Plasmid DNA can then be recoveredfrom the cells which score for inhibition, or alternatively,potentiation of acetylcholine induction, and the individual clonesfurther characterized.

An isolated mACHR-6 polypeptide, or a portion or fragment thereof, canbe used as an immunogen to generate antibodies that bind mACHR-6 usingstandard techniques for polyclonal and monoclonal antibody preparation.The full-length mACHR-6 polypeptide can be used or, alternatively, theinvention provides antigenic peptide fragments of mACHR-6 for use asimmunogens. The antigenic peptide of mACHR-6 comprises at least 8 aminoacid residues of the amino acid sequence shown in SEQ ID NO:2, 5, or 32and encompasses an epitope of mACHR-6 such that an antibody raisedagainst the peptide forms a specific immune complex with mACHR-6.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues. Preferred epitopes encompassed by the antigenicpeptide are regions of mACHR-6 that are located on the surface of thepolypeptide, e.g., hydrophilic regions.

An mACHR-6 immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed mACHR-6 polypeptide or achemically synthesized mACHR-6 peptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic mACHR-6 preparation induces a polyclonalanti-mACHR-6 antibody response.

Accordingly, another aspect of the invention pertains to anti-mACHR-6antibodies. The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds (immunoreacts with) an antigen, such as mACHR-6.Examples of immunologically active portions of immunoglobulin moleculesinclude F(ab) and F(ab′)₂ fragments which can be generated by treatingthe antibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind mACHR-6. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of mACHR-6. A monoclonal antibody composition thustypically displays a single binding affinity for a particular mACHR-6polypeptide with which it immunoreacts.

Polyclonal anti-mACHR-6 antibodies can be prepared as described above byimmunizing a suitable subject with an mACHR-6 immunogen. Theanti-mACHR-6 antibody titer in the immunized subject can be monitoredover time by standard techniques, such as with an enzyme linkedimmunosorbent assay (ELISA) using immobilized mACHR-6. If desired, theantibody molecules directed against mACHR-6 can be isolated from themammal (e.g., from the blood) and further purified by well knowntechniques, such as protein A chromatography to obtain the IgG fraction.At an appropriate time after immunization, e.g., when the anti-mACHR-6antibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al.(1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem.255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982)Int. J. Cancer 29:269-75), the more recent human B cell hybridomatechnique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridomatechnique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner(1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977)Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with an mACHR-6 immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds mACHR-6.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-mACHR-6 monoclonal antibody (see, e.g., G. Galfre et al. (1977)Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lemer,Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, citedsupra). Moreover, the ordinarily skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines can be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from ATCC®. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindmACHR-6, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-mACHR-6 antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with mACHR-6 to thereby isolateimmunoglobulin library members that bind mACHR-6. Kits for generatingand screening phage display libraries are commercially available (e.g.,the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01;and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol.Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram etal. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137;Barbas et al. (1991) PNAS 88:7978-7982; and McCafferty et al. Nature(1990) 348:552-554.

Additionally, recombinant anti-mACHR-6 antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.PCT International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)PNAS 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun etal. (1987) PNAS 84:214-218; Nishimura et al. (1987) Canc. Res.47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al.(1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985)Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S.Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan etal. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

An anti-mACHR-6 antibody (e.g., monoclonal antibody) can be used toisolate mACHR-6 by standard techniques, such as affinity chromatographyor immunoprecipitation. An anti-mACHR-6 antibody can facilitate thepurification of natural mACHR-6 from cells and of recombinantly producedmACHR-6 expressed in host cells. Moreover, an anti-mACHR-6 antibody canbe used to detect mACHR-6 polypeptide (e.g., in a cellular lysate orcell supernatant) in order to evaluate the abundance and pattern ofexpression of the mACHR-6 polypeptide or a fragment of an mACHR-6polypeptide. The detection of circulating fragments of an mACHR-6polypeptide can be used to identify mACHR-6 turnover in a subject.Anti-mACHR-6 antibodies can be used diagnostically to monitorpolypeptide levels in tissue as part of a clinical testing procedure,e.g., to, for example, determine the efficacy of a given treatmentregimen. Detection can be facilitated by coupling (i.e., physicallylinking) the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, 0-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

IV. Pharmaceutical Compositions

The mACHR-6 nucleic acid molecules, mACHR-6 polypeptides (particularlyfragments of mACHR-6), mACHR-6 modulators, and anti-mACHR-6 antibodies(also referred to herein as “active compounds”) of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject, e.g., a human. Such compositions typicallycomprise the nucleic acid molecule, polypeptide, modulator, or antibodyand a pharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, such media can be used in the compositions of theinvention. Supplementary active compounds can also be incorporated intothe compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., an mACHR-6 polypeptide or anti-mACHR-6 antibody) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) PNAS 91:3054-3057). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, polypeptides, polypeptide homologues,modulators, and antibodies described herein can be used in one or moreof the following methods: a) drug screening assays; b) diagnostic assaysparticularly in disease identification, allelic screening andpharmocogenetic testing; c) methods of treatment; d) pharmacogenomics;and e) monitoring of effects during clinical trials. An mACHR-6polypeptide of the invention has one or more of the activities describedherein and can thus be used to, for example, modulate an acetylcholineresponse in an acetylcholine responsive cell, for example by binding toacetylcholine or an mACHR-6 binding partner making it unavailable forbinding to the naturally present mACHR-6 polypeptide. The isolatednucleic acid molecules of the invention can be used to express mACHR-6polypeptide (e.g., via a recombinant expression vector in a host cell orin gene therapy applications), to detect mACHR-6 mRNA (e.g., in abiological sample) or a naturally occurring or recombinantly generatedgenetic mutation in an mACHR-6 gene, and to modulate mACHR-6 activity,as described further below. In addition, the mACHR-6 polypeptides can beused to screen drugs or compounds which modulate mACHR-6 polypeptideactivity as well as to treat disorders characterized by insufficientproduction of mACHR-6 polypeptide or production of mACHR-6 polypeptideforms which have decreased activity compared to wild type mACHR-6.Moreover, the anti-mACHR-6 antibodies of the invention can be used todetect and isolate an mACHR-6 polypeptide, particularly fragments ofmACHR-6 present in a biological sample, and to modulate mACHR-6polypeptide activity.

a. Drug Screening Assays:

The invention provides methods for identifying compounds or agents whichcan be used to treat disorders characterized by (or associated with)aberrant or abnormal mACHR-6 nucleic acid expression and/or mACHR-6polypeptide activity. These methods are also referred to herein as drugscreening assays and typically include the step of screening acandidate/test compound or agent to be an agonist or antagonist ofmACHR-6, and specifically for the ability to interact with (e.g., bindto) an mACHR-6 polypeptide, to modulate the interaction of an mACHR-6polypeptide and a target molecule, and/or to modulate mACHR-6 nucleicacid expression and/or mACHR-6 polypeptide activity. Candidate/testcompounds or agents which have one or more of these abilities can beused as drugs to treat disorders characterized by aberrant or abnormalmACHR-6 nucleic acid expression and/or mACHR-6 polypeptide activity.Candidate/test compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam, K. S. et al. (1991) Nature354:82-84; Houghten, R. et al. (1991) Nature 354:84-86) andcombinatorial chemistry-derived molecular libraries made of D- and/orL-configuration amino acids; 2) phosphopeptides (e.g., members of randomand partially degenerate, directed phosphopeptide libraries, see, e.g.,Songyang, Z. et al. (1993) Cell 72:767-778); 3) antibodies (e.g.,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and singlechain antibodies as well as Fab, F(ab′)₂, Fab expression libraryfragments, and epitope-binding fragments of antibodies); and 4) smallorganic and inorganic molecules (e.g., molecules obtained fromcombinatorial and natural product libraries).

In one embodiment, the invention provides assays for screeningcandidate/test compounds which interact with (e.g., bind to) mACHR-6polypeptide. Typically, the assays are recombinant cell based orcell-free assays which include the steps of combining an mACHR-6polypeptide or a bioactive fragment thereof, and a candidate/testcompound, e.g., under conditions which allow for interaction of (e.g.,binding of) the candidate/test compound to the mACHR-6 polypeptide orfragment thereof to form a complex, and detecting the formation of acomplex, in which the ability of the candidate compound to interact with(e.g., bind to) the mACHR-6 polypeptide or fragment thereof is indicatedby the presence of the candidate compound in the complex. Formation ofcomplexes between the mACHR-6 polypeptide and the candidate compound canbe quantitated, for example, using standard immunoassays.

In another embodiment, the invention provides screening assays toidentify candidate/test compounds which modulate (e.g., stimulate orinhibit) the interaction (and most likely mACHR-6 activity as well)between an mACHR-6 polypeptide and a molecule (target molecule) withwhich the mACHR-6 polypeptide normally interacts. Examples of suchtarget molecules include polypeptides in the same signaling path as themACHR-6 polypeptide, e.g., polypeptides which may function upstream(including both stimulators and inhibitors of activity) or downstream ofthe mACHR-6 polypeptide in, for example, a cognitive function signalingpathway or in a pathway involving mACHR-6 activity, e.g., a G protein orother interactor involved in phosphatidylinositol turnover and/orphospholipase C activation. Typically, the assays are recombinant cellbased or cell-free assays which include the steps of combining a cellexpressing an mACHR-6 polypeptide, or a bioactive fragment thereof, anmACHR-6 target molecule (e.g., an mACHR-6 ligand) and a candidate/testcompound, e.g., under conditions wherein but for the presence of thecandidate compound, the mACHR-6 polypeptide or biologically activeportion thereof interacts with (e.g., binds to) the target molecule, anddetecting the formation of a complex which includes the mACHR-6polypeptide and the target molecule or detecting theinteraction/reaction of the mACHR-6 polypeptide and the target molecule.Detection of complex formation can include direct quantitation of thecomplex by, for example, measuring inductive effects of the mACHR-6polypeptide. A statistically significant change, such as a decrease, inthe interaction of the mACHR-6 and target molecule (e.g., in theformation of a complex between the mACHR-6 and the target molecule) inthe presence of a candidate compound (relative to what is detected inthe absence of the candidate compound) is indicative of a modulation(e.g., stimulation or inhibition) of the interaction between the mACHR-6polypeptide and the target molecule. Modulation of the formation ofcomplexes between the mACHR-6 polypeptide and the target molecule can bequantitated using, for example, an immunoassay.

To perform cell free drug screening assays, it is desirable toimmobilize either mACHR-6 or its target molecule to facilitateseparation of complexes from uncomplexed forms of one or both of thepolypeptides, as well as to accommodate automation of the assay.Interaction (e.g., binding of) of mACHR-6 to a target molecule, in thepresence and absence of a candidate compound, can be accomplished in anyvessel suitable for containing the reactants. Examples of such vesselsinclude microtitre plates, test tubes, and micro-centrifuge tubes. Inone embodiment, a fusion polypeptide can be provided which adds a domainthat allows the polypeptide to be bound to a matrix. For example,glutathione-S-transferase/mACHR-6 fusion polypeptides can be adsorbedonto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofmACHR-6-binding polypeptide found in the bead fraction quantitated fromthe gel using standard electrophoretic techniques.

Other techniques for immobilizing polypeptides on matrices can also beused in the drug screening assays of the invention. For example, eithermACHR-6 or its target molecule can be immobilized utilizing conjugationof biotin and streptavidin. Biotinylated mACHR-6 molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive withmACHR-6 but which do not interfere with binding of the polypeptide toits target molecule can be derivatized to the wells of the plate, andmACHR-6 trapped in the wells by antibody conjugation. As describedabove, preparations of an mACHR-6 -binding polypeptide and a candidatecompound are incubated in the mACHR-6 -presenting wells of the plate,and the amount of complex trapped in the well can be quantitated.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the mACHR-6 target molecule, orwhich are reactive with mACHR-6 polypeptide and compete with the targetmolecule; as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the target molecule.

In yet another embodiment, the invention provides a method foridentifying a compound (e.g., a screening assay) capable of use in thetreatment of a disorder characterized by (or associated with) aberrantor abnormal mACHR-6 nucleic acid expression or mACHR-6 polypeptideactivity. This method typically includes the step of assaying theability of the compound or agent to modulate the expression of themACHR-6 nucleic acid or the activity of the mACHR-6 polypeptide therebyidentifying a compound for treating a disorder characterized by aberrantor abnormal mACHR-6 nucleic acid expression or mACHR-6 polypeptideactivity. Disorders characterized by aberrant or abnormal mACHR-6nucleic acid expression or mACHR-6 polypeptide activity are describedherein. Methods for assaying the ability of the compound or agent tomodulate the expression of the mACHR-6 nucleic acid or activity of themACHR-6 polypeptide are typically cell-based assays. For example, cellswhich are sensitive to ligands which transduce signals via a pathwayinvolving mACHR-6 can be induced to overexpress an mACHR-6 polypeptidein the presence and absence of a candidate compound. Candidate compoundswhich produce a statistically significant change in mACHR-6 -dependentresponses (either stimulation or inhibition) can be identified. In oneembodiment, expression of the mACHR-6 nucleic acid or activity of anmACHR-6 polypeptide is modulated in cells and the effects of candidatecompounds on the readout of interest (such as phosphatidylinositolturnover) are measured. For example, the expression of genes which areup- or down-regulated in response to an mACHR-6-dependent signal cascadecan be assayed. In preferred embodiments, the regulatory regions of suchgenes, e.g., the 5′ flanking promoter and enhancer regions, are operablylinked to a detectable marker (such as luciferase) which encodes a geneproduct that can be readily detected. Phosphorylation of mACHR-6 ormACHR-6 target molecules can also be measured, for example, byimmunoblotting.

Alternatively, modulators of mACHR-6 expression (e.g., compounds whichcan be used to treat a disorder characterized by aberrant or abnormalmACHR-6 nucleic acid expression or mACHR-6 polypeptide activity) can beidentified in a method wherein a cell is contacted with a candidatecompound and the expression of mACHR-6 mRNA or polypeptide in the cellis determined. The level of expression of mACHR-6 mRNA or polypeptide inthe presence of the candidate compound is compared to the level ofexpression of mACHR-6 mRNA or polypeptide in the absence of thecandidate compound. The candidate compound can then be identified as amodulator of mACHR-6 nucleic acid expression based on this comparisonand be used to treat a disorder characterized by aberrant mACHR-6nucleic acid expression. For example, when expression of mACHR-6 mRNA orpolypeptide is greater (statistically significantly greater) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of mACHR-6 nucleic acidexpression. Alternatively, when mACHR-6 nucleic acid expression is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of mACHR-6 nucleic acid expression. The level of mACHR-6nucleic acid expression in the cells can be determined by methodsdescribed herein for detecting mACHR-6 mRNA or polypeptide.

In yet another aspect of the invention, the mACHR-6 polypeptides, orfragments thereof, can be used as “bait proteins” in a two-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO 94/10300), to identify other proteins, whichbind to or interact with mACHR-6 (“mACHR-6-binding proteins” or“mACHR-6-bp”) and modulate mACHR-6 polypeptide activity. SuchmACHR-6-binding proteins are also likely to be involved in thepropagation of signals by the mACHR-6 polypeptides as, for example,upstream or downstream elements of the mACHR-6 pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Bartel, P. et al. “Using the Two-Hybrid System toDetect Protein-Protein Interactions” in Cellular Interactions inDevelopment: A Practical Approach, Hartley, D. A. ed. (Oxford UniversityPress, Oxford, 1993) pp. 153-179. Briefly, the assay utilizes twodifferent DNA constructs. In one construct, the gene that codes formACHR-6 is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming anmACHR-6-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with mACHR-6.

Modulators of mACHR-6 polypeptide activity and/or mACHR-6 nucleic acidexpression identified according to these drug screening assays can beused to treat, for example, nervous system disorders, smooth musclerelated disorders, cardiac muscle related disorders, and gland relateddisorders. These methods of treatment include the steps of administeringthe modulators of mACHR-6 polypeptide activity and/or nucleic acidexpression, e.g., in a pharmaceutical composition as described insubsection IV above, to a subject in need of such treatment, e.g., asubject with a disorder described herein.

b. Diagnostic Assays:

The invention further provides a method for detecting the presence ofmACHR-6, or fragment thereof, in a biological sample. The methodinvolves contacting the biological sample with a compound or an agentcapable of detecting mACHR-6 polypeptide or mRNA such that the presenceof mACHR-6 is detected in the biological sample. A preferred agent fordetecting mACHR-6 mRNA is a labeled or labelable nucleic acid probecapable of hybridizing to mACHR-6 MRNA. The nucleic acid probe can be,for example, the full-length mACHR-6 cDNA of SEQ ID NO:1, 4, or 31, or aportion thereof, such as an oligonucleotide of at least 15, 30, 50, 100,250 or 500 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to mACHR-6 mRNA. A preferred agentfor detecting mACHR-6 polypeptide is a labeled or labelable antibodycapable of binding to mACHR-6 polypeptide. Antibodies can be polyclonal,or more preferably, monoclonal. An intact antibody, or a fragmentthereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled orlabelable”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. That is, the detection method of the inventioncan be used to detect mACHR-6 mRNA or polypeptide in a biological samplein vitro as well as in vivo. For example, in vitro techniques fordetection of mACHR-6 mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of mACHR-6 polypeptideinclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. Alternatively, mACHR-6polypeptide can be detected in vivo in a subject by introducing into thesubject a labeled anti-mACHR-6 antibody. For example, the antibody canbe labeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques. Particularlyuseful are methods which detect the allelic variant of mACHR-6 expressedin a subject and methods which detect fragments of an mACHR-6polypeptide in a sample.

The invention also encompasses kits for detecting the presence ofmACHR-6 in a biological sample. For example, the kit can comprise alabeled or labelable compound or agent capable of detecting mACHR-6polypeptide or mRNA in a biological sample; means for determining theamount of mACHR-6 in the sample; and means for comparing the amount ofmACHR-6 in the sample with a standard. The compound or agent can bepackaged in a suitable container. The kit can further compriseinstructions for using the kit to detect mACHR-6 mRNA or polypeptide.

The methods of the invention can also be used to detect naturallyoccurring genetic mutations in an mACHR-6 gene, thereby determining if asubject with the mutated gene is at risk for a disorder characterized byaberrant or abnormal mACHR-6 nucleic acid expression or mACHR-6polypeptide activity as described herein. In preferred embodiments, themethods include detecting, in a sample of cells from the subject, thepresence or absence of a genetic mutation characterized by at least oneof an alteration affecting the integrity of a gene encoding an mACHR-6polypeptide, or the misexpression of the mACHR-6 gene. For example, suchgenetic mutations can be detected by ascertaining the existence of atleast one of 1) a deletion of one or more nucleotides from an mACHR-6gene; 2) an addition of one or more nucleotides to an mACHR-6 gene; 3) asubstitution of one or more nucleotides of an mACHR-6 gene, 4) achromosomal rearrangement of an mACHR-6 gene; 5) an alteration in thelevel of a messenger RNA transcript of an mACHR-6 gene, 6) aberrantmodification of an mACHR-6 gene, such as of the methylation pattern ofthe genomic DNA, 7) the presence of a non-wild type splicing pattern ofa messenger RNA transcript of an mACHR-6 gene, 8) a non-wild type levelof an mACHR-6-polypeptide, 9) allelic loss of an mACHR-6 gene, and 10)inappropriate post-translational modification of an mACHR-6-polypeptide.As described herein, there are a large number of assay techniques knownin the art which can be used for detecting mutations in an mACHR-6 gene.

In certain embodiments, detection of the mutation involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS91:360-364), the latter of which can be particularly useful fordetecting point mutations in the mACHR-6-gene (see Abravaya et al.(1995) Nucleic Acids Res .23:675-682). This method can include the stepsof collecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to an mACHR-6 gene under conditions such that hybridizationand amplification of the mACHR-6-gene (if present) occurs, and detectingthe presence or absence of an amplification product, or detecting thesize of the amplification product and comparing the length to a controlsample.

In an alternative embodiment, mutations in an mACHR-6 gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the mACHR-6 gene anddetect mutations by comparing the sequence of the sample mACHR-6 withthe corresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxim andGilbert ((1977) PNAS 74:560) or Sanger ((1977) PNAS 74:5463). A varietyof automated sequencing procedures can be utilized when performing thediagnostic assays ((1995) Biotechniques 19:448), including sequencing bymass spectrometry (see, e.g., PCT International Publication No. WO94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffinet al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in the mACHR-6 gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al. (1985)Science 230:1242); Cotton et al. (1988) PNAS 85:4397; Saleeba et al.(1992) Meth. Enzymol. 217:286-295), electrophoretic mobility of mutantand wild type nucleic acid is compared (Orita et al. (1989) PNAS86:2766; Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) GenetAnal Tech Appl 9:73-79), and movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (Myers et al (1985) Nature313:495). Examples of other techniques for detecting point mutationsinclude, selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

c. Methods of Treatment

Another aspect of the invention pertains to methods for treating asubject, e.g., a human, having a disease or disorder characterized by(or associated with) aberrant or abnormal mACHR-6 nucleic acidexpression and/or mACHR-6 polypeptide activity. These methods includethe step of administering an mACHR-6 modulator (agonist or antagonist)to the subject such that treatment occurs. The language “aberrant orabnormal mACHR-6 expression” refers to expression of a non-wild-typemACHR-6 polypeptide or a non-wild-type level of expression of an mACHR-6polypeptide. Aberrant or abnormal mACHR-6 activity refers to anon-wild-type mACHR-6 activity or a non-wild-type level of mACHR-6activity. As the mACHR-6 polypeptide is involved in a pathway involvingmodulation of neurotransmitter, e.g., acetylcholine, release; modulationof smooth muscle contraction; modulation of cardiac muscle contraction;and modulation of gland, e.g., exocrine gland function, aberrant orabnormal mACHR-6 activity or expression interferes with the normalneurotransmitter, e.g., acetylcholine, release; normal smooth muscle;and cardiac muscle contraction; and normal gland, e.g., exocrine glandfunction. Non-limiting examples of disorders or diseases characterizedby or associated with abnormal or aberrant mACHR-6 activity orexpression include nervous system related disorders, e.g., centralnervous system related disorders. Examples of nervous system relateddisorders include cognitive disorders, e.g., memory and learningdisorders, such as amnesia, apraxia, agnosia, amnestic dysnomia,amnestic spatial disorientation, Kluver-Bucy syndrome, Alzheimer'srelated memory loss (Eglen R. M. (1996) Pharmacol. and Toxicol.78(2):59-68; Perry E. K. (1995) Brain and Cognition 28(3):240-58) andlearning disability; disorders affecting consciousness, e.g., visualhallucinations, perceptual disturbances, or delerium associated withLewy body dementia; schitzo-effective disorders (Dean B. (1996) Mol.Psychiatry 1(1):54-8), schizophrenia with mood swings (Bymaster F. P.(1997) J. Clin. Psychiatry 58 (suppl.10):28-36; Yeomans J. S. (1995)Neuropharmacol. 12(1):3-16; Reimann D. (1994) J. Psychiatric Res.28(3):195-210), depressive illness (primary or secondary); affectivedisorders (Janowsky D. S. (1994) Am. J Med. Genetics 54(4):335-44);sleep disorders (Kimura F. (1997) J. Neurophysiol. 77(2):709-16), e.g.,REM sleep abnormalities in patients suffering from, for example,depression (Riemann D. (1994) J. Psychosomatic Res. 38 Suppl. 1:15-25;Bourgin P. (1995) Neuroreport 6(3): 532-6), paradoxical sleepabnormalities (Sakai K. (1997) Eur. J. Neuroscience 9(3):415-23),sleep-wakefulness, and body temperature or respiratory depressionabnormalities during sleep (Shuman S. L. (1995) Am. J. Physiol. 269(2 Pt2):R308-17; Mallick B. N. (1997) Brain Res. 750(1-2):311-7). Otherexamples of nervous system related disorders include disorders affectingpain generation mechanisms, e.g., pain related to irritable bowelsyndrome (Mitch C. H. (1997) J. Med. Chem. 40(4):538-46; Shannon H. E.(1997) J. Pharmac. and Exp. Therapeutics 281(2):884-94; Bouaziz H.(1995) Anesthesia and Analgesia 80(6):1140-4; or Guimaraes A. P. (1994)Brain Res. 647(2):220-30) or chest pain; movement disorders (Monassi C.R. (1997) Physiol. and Behav. 62(1):53-9), e.g., Parkinson's diseaserelated movement disorders (Finn M. (1997) Pharmacol Biochem. & Behavior57(1-2):243-9; Mayorga A. J. (1997) Pharmacol. Biochem. & Behavior56(2):273-9); eating disorders, e.g., insulin hypersecretion relatedobesity (Maccario M. (1997) J. Endocrinol. Invest. 20(1):8-12;Premawardhana L. D. (1994) Clin. Endocrinol. 40(5): 617-21); or drinkingdisorders, e.g., diabetic polydipsia (Murzi E. (1997) Brain Res.752(1-2):184-8; Yang X. (1994) Pharmacol. Biochem. & Behavior49(1):1-6). Yet further examples of disorders or diseases characterizedby or associated with abnormal or aberrant mACHR-6 activity orexpression include smooth muscle related disorders such as irritablebowel syndrome, diverticular disease, urinary incontinence, oesophagealachalasia, or chronic obstructive airways disease; heart muscle relateddisorders such as pathologic bradycardia or tachycardia, arrhythmia,flutter or fibrillation; or gland related disorders such as xerostomia,or diabetes mellitus. The terms “treating” or “treatment”, as usedherein, refer to reduction or alleviation of at least one adverse effector symptom of a disorder or disease, e.g., a disorder or diseasecharacterized by or associated with abnormal or aberrant mACHR-6polypeptide activity or mACHR-6 nucleic acid expression.

As used herein, an mACHR-6 modulator is a molecule which can modulatemACHR-6 nucleic acid expression and/or mACHR-6 polypeptide activity. Forexample, an mACHR-6 modulator can modulate, e.g., upregulate(activate/agonize) or downregulate (suppress/antagonize), mACHR-6nucleic acid expression. In another example, an mACHR-6 modulator canmodulate (e.g., stimulate/agonize or inhibit/antagonize) mACHR-6polypeptide activity. If it is desirable to treat a disorder or diseasecharacterized by (or associated with) aberrant or abnormal(non-wild-type) mACHR-6 nucleic acid expression and/or mACHR-6polypeptide activity by inhibiting mACHR-6 nucleic acid expression, anmACHR-6 modulator can be an antisense molecule, e.g., a ribozyme, asdescribed herein. Examples of antisense molecules which can be used toinhibit mACHR-6 nucleic acid expression include antisense moleculeswhich are complementary to a portion of the 5′ untranslated region ofSEQ ID NO:1 which also includes the start codon and antisense moleculeswhich are complementary to a portion of the 3′ untranslated region ofSEQ ID NO:1, 4, or 31. An example of an antisense molecule which iscomplementary to a portion of the 5′ untranslated region of SEQ ID NO:1and which also includes the start codon is a nucleic acid molecule whichincludes nucleotides which are complementary to nucleotides 280 to 296of SEQ ID NO:1. This antisense molecule has the following nucleotidesequence: 5′ CCTGCGGGGCCATGGAG 3′ (SEQ ID NO:21). An example of anantisense molecule which is complementary to a portion of the 3′untranslated region of SEQ ID NO:1 is a nucleic acid molecule whichincludes nucleotides which are complementary to nucleotides 1629 to 1645of SEQ ID NO:1. This antisense molecule has the following sequence: 5′GTGGCCCACCAGAGCCT 3′ (SEQ ID NO:22). An additional example of anantisense molecule which is complementary to a portion of the 3′untranslated region of SEQ ID NO:1 is a nucleic acid molecule whichincludes nucleotides which are complementary to nucleotides 1650 to 1666of SEQ ID NO:1. This antisense molecule has the following sequence: 5′CAGCCACGCCTCTCTCA 3′ (SEQ ID NO:23). An example of an antisense moleculewhich is complementary to a portion of the 5′ untranslated region of SEQID NO:4 and which also includes the start codon, is a nucleic acidmolecule which includes nucleotides which are complementary tonucleotides 766 to 783 of SEQ ID NO:4. This antisense molecule has thefollowing nucleotide sequence: 5′ GCCTGCTGGGCCATGGAG 3′ (SEQ ID NO:24).An example of an antisense molecule which is complementary to a portionof the 3′ untranslated region of SEQ ID NO:4 is a nucleic acid moleculewhich includes nucleotides which are complementary to nucleotides 2113to 2128 of SEQ ID NO:4. This antisense molecule has the followingsequence: 5′ TGAGCAGCTGCCCCAC 3′ (SEQ ID NO:25). An additional exampleof an antisense molecule which is complementary to a portion of the 3′untranslated region of SEQ ID NO:4 is a nucleic acid molecule whichincludes nucleotides which are complementary to nucleotides 2133 to 2148of SEQ ID NO:4. This antisense molecule has the following sequence: 5′CTGAGGCCAGGCCCTT 3′ (SEQ ID NO:26).

An mACHR-6 modulator which inhibits mACHR-6 nucleic acid expression canalso be a small molecule or other drug, e.g., a small molecule or drugidentified using the screening assays described herein, which inhibitsmACHR-6 nucleic acid expression. If it is desirable to treat a diseaseor disorder characterized by (or associated with) aberrant or abnormal(non-wild-type) mACHR-6 nucleic acid expression and/or mACHR-6polypeptide activity by stimulating mACHR-6 nucleic acid expression, anmACHR-6 modulator can be, for example, a nucleic acid molecule encodingmACHR-6 (e.g., a nucleic acid molecule comprising a nucleotide sequencehomologous to the nucleotide sequence of SEQ ID NO:1, 4, or 31) or asmall molecule or other drug, e.g., a small molecule (peptide) or drugidentified using the screening assays described herein, which stimulatesmACHR-6 nucleic acid expression.

Alternatively, if it is desirable to treat a disease or disordercharacterized by (or associated with) aberrant or abnormal(non-wild-type) mACHR-6 nucleic acid expression and/or mACHR-6polypeptide activity by inhibiting mACHR-6 polypeptide activity, anmACHR-6 modulator can be an anti-mACHR-6 antibody or a small molecule orother drug, e.g., a small molecule or drug identified using thescreening assays described herein, which inhibits mACHR-6 polypeptideactivity. If it is desirable to treat a disease or disordercharacterized by (or associated with) aberrant or abnormal(non-wild-type) mACHR-6 nucleic acid expression and/or mACHR-6polypeptide activity by stimulating mACHR-6 polypeptide activity, anmACHR-6 modulator can be an active mACHR-6 polypeptide or portionthereof (e.g., an mACHR-6 polypeptide or portion thereof having an aminoacid sequence which is homologous to the amino acid sequence of SEQ IDNO:2, 5, or 32 or a portion thereof) or a small molecule or other drug,e.g., a small molecule or drug identified using the screening assaysdescribed herein, which stimulates mACHR-6 polypeptide activity.

Other aspects of the invention pertain to methods for modulating a cellassociated activity. These methods include contacting the cell with anagent (or a composition which includes an effective amount of an agent)which modulates mACHR-6 polypeptide activity or mACHR-6 nucleic acidexpression such that a cell associated activity is altered relative to acell associated activity (for example, phosphatidylinositol metabolism)of the cell in the absence of the agent. As used herein, “a cellassociated activity” refers to a normal or abnormal activity or functionof a cell. Examples of cell associated activities includephosphatidylinositol turnover, production or secretion of molecules,such as proteins, contraction, proliferation, migration,differentiation, and cell survival. In a preferred embodiment, the cellis neural cell of the brain, e.g., a hippocampal cell. The term“altered” as used herein refers to a change, e.g., an increase ordecrease, of a cell associated activity particularlyphosphatidylinositol turnover and phospholipase C activation. In oneembodiment, the agent stimulates mACHR-6 polypeptide activity or mACHR-6nucleic acid expression. Examples of such stimulatory agents include anactive mACHR-6 polypeptide, a nucleic acid molecule encoding mACHR-6that has been introduced into the cell, and a modulatory agent whichstimulates mACHR-6 polypeptide activity or mACHR-6 nucleic acidexpression and which is identified using the drug screening assaysdescribed herein. In another embodiment, the agent inhibits mACHR-6polypeptide activity or mACHR-6 nucleic acid expression. Examples ofsuch inhibitory agents include an antisense mACHR-6 nucleic acidmolecule, an anti-mACHR-6 antibody, and a modulatory agent whichinhibits mACHR-6 polypeptide activity or mACHR-6 nucleic acid expressionand which is identified using the drug screening assays describedherein. These modulatory methods can be performed in vitro (e.g., byculturing the cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject). In a preferred embodiment, themodulatory methods are performed in vivo, i.e., the cell is presentwithin a subject, e.g., a mammal, e.g., a human, and the subject has adisorder or disease characterized by or associated with abnormal oraberrant mACHR-6 polypeptide activity or mACHR-6 nucleic acidexpression.

A nucleic acid molecule, a polypeptide, an mACHR-6 modulator, a compoundetc. used in the methods of treatment can be incorporated into anappropriate pharmaceutical composition described herein and administeredto the subject through a route which allows the molecule, polypeptide,modulator, or compound etc. to perform its intended function. Examplesof routes of administration are also described herein under subsectionIV.

d. Pharmacogenomics

Test/candidate compounds, or modulators which have a stimulatory orinhibitory effect on mACHR-6 activity (e.g., mACHR-6 gene expression) asidentified by a screening assay described herein can be administered toindividuals to treat (prophylactically or therapeutically) disorders(e.g., CNS disorders) associated with aberrant mACHR-6 activity. Inconjunction with such treatment, the pharmacogenomics (i.e., the studyof the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) of the individualmay be considered. Differences in metabolism of therapeutics can lead tosevere toxicity or therapeutic failure by altering the relation betweendose and blood concentration of the pharmacologically active drug. Thus,the pharmacogenomics of the individual permit the selection of effectivecompounds (e.g., drugs) for prophylactic or therapeutic treatments basedon a consideration of the individual's genotype. Such pharmacogenomicscan further be used to determine appropriate dosages and therapeuticregimens. Accordingly, the activity of mACHR-6 polypeptide, expressionof mACHR-6 nucleic acid, or mutation content of mACHR-6 genes in anindividual can be determined to thereby select appropriate compound(s)for therapeutic or prophylactic treatment of the individual.

Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (1996) Clin. Exp.Pharmacol Physiol. 23(10-11) :983-985 and Linder, M. W. (1997) Clin.Chem. 43(2):254-266. In general, two types of pharmacogenetic conditionscan be differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Thus, the activity of mACHR-6 polypeptide, expression of mACHR-6 nucleicacid, or mutation content of mACHR-6 genes in an individual can bedetermined to thereby select appropriate agent(s) for therapeutic orprophylactic treatment of a subject. In addition, pharmacogeneticstudies can be used to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of a subject's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith an mACHR-6 modulator, such as a modulator identified by one of theexemplary screening assays described herein.

e. Monitoring of Effects During Clinical Trials

Monitoring the influence of compounds (e.g., drugs) on the expression oractivity of mACHR-6 (e.g., the ability to modulate the effects ofacetylcholine on acetylcholine responsive cells) can be applied not onlyin basic drug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay, as describedherein, to increase mACHR-6 gene expression, polypeptide levels, orup-regulate mACHR-6 activity, can be monitored in clinical trails ofsubjects exhibiting decreased mACHR-6 gene expression, polypeptidelevels, or down-regulated mACHR-6 activity. Alternatively, theeffectiveness of an agent, determined by a screening assay, to decreasemACHR-6 gene expression, polypeptide levels, or down-regulate mACHR-6activity, can be monitored in clinical trails of subjects exhibitingincreased mACHR-6 gene expression, polypeptide levels, or up-regulatedmACHR-6 activity. In such clinical trials, the expression or activity ofmACHR-6 and, preferably, other genes which have been implicated in, forexample, a nervous system related disorder can be used as a “read out”or markers of the acetylcholine responsiveness of a particular cell.

For example, and not by way of limitation, genes, including mACHR-6,which are modulated in cells by treatment with a compound (e.g., drug orsmall molecule) which modulates mACHR-6 activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of compounds on CNS disorders, for example, in a clinicaltrial, cells can be isolated and RNA prepared and analyzed for thelevels of expression of mACHR-6 and other genes implicated in thedisorder. The levels of gene expression (i.e., a gene expressionpattern) can be quantified by Northern blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount ofpolypeptide produced, by one of the methods described herein, or bymeasuring the levels of activity of mACHR-6 or other genes. In this way,the gene expression pattern can serve as a marker, indicative of thephysiological response of the cells to the compound. Accordingly, thisresponse state may be determined before, and at various points during,treatment of the individual with the compound.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with a compound(e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide,nucleic acid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe compound; (ii) detecting the level of expression of an mACHR-6polypeptide, mRNA, or genomic DNA in the preadministration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression or activity of the mACHR-6polypeptide, mRNA, or genomic DNA in the post-administration samples;(v) comparing the level of expression or activity of the mACHR-6polypeptide, mRNA, or genomic DNA in the pre-administration sample withthe mACHR-6 polypeptide, mRNA, or genomic DNA in the post administrationsample or samples; and (vi) altering the administration of the compoundto the subject accordingly. For example, increased administration of thecompound may be desirable to increase the expression or activity ofmACHR-6 to higher levels than detected, i.e., to increase theeffectiveness of the agent. Alternatively, decreased administration ofthe agent may be desirable to decrease expression or activity of mACHR-6to lower levels than detected, i.e. to decrease the effectiveness of thecompound.

VI. Uses of Partial mACHR-6 Sequences

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(a) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (b) identify an individual froma minute biological sample (tissue typing); and (c) aid in forensicidentification of a biological sample. These applications are describedin the subsections below.

a. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the mACHR-6, sequences, described herein, canbe used to map the location of the mACHR-6 gene, respectively, on achromosome. The mapping of the mACHR-6 sequence to chromosomes is animportant first step in correlating these sequence with genes associatedwith disease.

Briefly, the mACHR-6 gene can be mapped to a chromosome by preparing PCRprimers (preferably 15-25 bp in length) from the mACHR-6 sequence.Computer analysis of the mACHR-6, sequence can be used to rapidly selectprimers that do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers can then be usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the mACHR-6 sequence will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media, panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes. (D'Eustachio P. et al. (1983)Science 220:919-924). Somatic cell hybrids containing only fragments ofhuman chromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the mACHR-6sequence to design oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes. Othermapping strategies which can similarly be used to map a mACHR-6 sequenceto its chromosome include in situ hybridization (described in Fan, Y. etal. (1990) PNAS, 87:6223-27), pre-screening with labeled flow-sortedchromosomes, and pre-selection by hybridization to chromosome specificcDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical likecolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York, 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data (such data are found, for example, in V. McKusick,Mendelian Inheritance in Man, available on-line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddisease, mapped to the same chromosomal region, can then be identifiedthrough linkage analysis (co-inheritance of physically adjacent genes),described in, for example, Egeland, J. et al. (1987) Nature,325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the mACHR-6 gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

b. Tissue Typing

The mACHR-6 sequences of the present invention can also be used toidentify individuals from minute biological samples. The United Statesmilitary, for example, is considering the use of restriction fragmentlength polymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the mACHR-6 sequences described herein can be used toprepare two PCR primers from the 5′ and 3′ ends of the sequences. Theseprimers can then be used to amplify an individual's DNA and subsequentlysequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The mACHR-6 sequences of the invention uniquely represent portions ofthe human genome. Allelic variation occurs to some degree in the codingregions of these sequences, and to a greater degree in the noncodingregions. It is estimated that allelic variation between individualhumans occurs with a frequency of about once per each 500 bases. Each ofthe sequences described herein can, to some degree, be used as astandard against which DNA from an individual can be compared foridentification purposes. Because greater numbers of polymorphisms occurin the noncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NOs:1, 4, and 31, cancomfortably provide positive individual identification with a panel ofperhaps 10 to 1,000 primers which each yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences, such as those inSEQ ID NOs:3, 6, and 33, are used, a more appropriate number of primersfor positive individual identification would be 500-2,000.

If a panel of reagents from mACHR-6 sequences described herein is usedto generate a unique identification database for an individual, thosesame reagents can later be used to identify tissue from that individual.Using the unique identification database, positive identification of theindividual, living or dead, can be made from extremely small tissuesamples.

c. Use of Partial mACHR-6 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As described above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NOs:1, 4, and 31 areparticularly appropriate for this use as greater numbers ofpolymorphisms occur in the noncoding regions, making it easier todifferentiate individuals using this technique. Examples ofpolynucleotide reagents include the mACHR-6 sequences or portionsthereof, e.g., fragments derived from the noncoding regions of SEQ IDNOs:1, 4, and 31, having a length of at least 20 bases, preferably atleast 30 bases.

The mACHR-6 sequences described herein can further be used to providepolynucleotide reagents, e.g., labeled or labelable probes which can beused in, for example, an in situ hybridization technique, to identify aspecific tissue, e.g., brain tissue. This can be very useful in caseswhere a forensic pathologist is presented with a tissue of unknownorigin. Panels of such mACHR-6 probes can be used to identify tissue byspecies and/or by organ type.

In a similar fashion, these reagents, e.g., mACHR-6 primers or probescan be used to screen tissue culture for contamination (i.e. screen forthe presence of a mixture of different types of cells in a culture).

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patent applications, patents, and published patent applications citedthroughout this application are hereby incorporated by reference.

EXAMPLES Example 1 Identification of Rat and Human mACHR-6 cDNA

In this example, mACHR-6 nucleic acid molecules were identified byscreening appropriate cDNA libraries. More specifically, a rat frontalcortex oligo dT-primed cDNA library was plated out and colonies pickedinto 96 well plates. The colonies were cultured, plasmids were preparedfrom each well, and the 5′ end of each insert sequenced. After automated“trimming” of non-insert sequences, the nucleotide sequences werecompared against the public protein databases using the BLAST sequencecomparison program (BLASTN1.3MP, Altschul et al. (1990) J. Mol. Biol.215:403). Upon review of the results from this sequence comparison, asingle clone was identified, designated 84g5, whose highest similaritywas with the rat muscarinic acetylcholine receptor M1 (mACHR M1;GenBank™ Accession Number P08482). The clone containing this sequencewas recovered from the 96 well plate, plasmid was prepared usingstandard methods and the insert fully sequenced using standard“contigging” techniques. A repeat BLAST analysis using the entire insertsequence once again showed that the sequence in the protein databasewith the greatest similarity corresponded to GenBank™ Accession NumberP08482. This sequence and the insert sequence were compared using theGAP program in the GCG software package using a gap weight of 5.000 anda length weight of 0.100. The results showed a 27.97% identity and49.01% similarity between the two sequences with the insertion of 4 gapsfor optimized sequence alignment. The alignment indicated that the 84g5clone does not extend fully across the P08482 sequence, apparentlymissing approximately 30 amino acid residues at the N-terminal region ofthe molecule. A probe spanning residues 143-249 of SEQ ID NO:31 was thenused to re-screen the same frontal cortex library. This resulted in theindentification of the full length rat mACHR-6 sequence shown in SEQ IDNO:4. BLAST analysis of public nucleotide databases revealed noequivalent human sequences. Only a single mouse EST was identified(GenBank™ Accession Number AA118949) which is similar to the 84g5 clonebetween residues 1101 and 1650.

The human mACHR-6 nucleic acid molecule was identified by screening ahuman cerebellum cDNA library using a Nci I/Not I restriction fragmentof the rat cDNA as a probe. BLAST analysis of protein and nucleic aciddatabases in the public domain again showed that the mACHR-6 nucleicacid molecule is most similar to mACHR M1 sequences. The alignments alsorevealed that mAChR-6 nucleic acid molecule encodes a full length mACHRpolypeptide.

Example 2 Tissue Expression of the mACHR-6 Gene

Northern Analysis Using RNA from Human and Rat Tissue

Human brain multiple tissue northern (MTN) blots, human MTN I, II andIII blots, and rat MTN blots (Clontech, Palo Alto, Calif.), containing 2g of poly A+ RNA per lane were probed with the rat mACHR-6 nucleotidesequence (Nci I/Not I restriction fragment). The filters wereprehybridized in 10 ml of Express Hyb hybridization solution (Clontech,Palo Alto, Calif.) at 68° C. for 1 hour, after which 100 ng of ³²Plabeled probe was added. The probe was generated using the StratagenePrime-It kit, Catalog Number 300392 (Clontech, Palo Alto, Calif.).Hybridization was allowed to proceed at 68° C. for approximately 2hours. The filters were washed in a 0.05% SDS/2×SSC solution for 15minutes at room temperature and then twice with a 0.1% SDS/0.1×SSCsolution for 20 minutes at 50° C. and then exposed to autoradiographyfilm overnight at −80° C. with one screen. The human tissues testedincluded: heart, brain (regions of the brain tested included cerebellum,corpus callosum, cerebral cortex, medulla, occipital pole, frontal lobe,temporal lobe, putamen, amygdala, caudate nucleus, hippocampus,substantia nigra, subthalamic nucleus and thalamus), placenta, lung,liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate,testis, ovary, small intestine, colon, peripheral blood leukocyte,stomach, thyroid, spinal cord, lymph node, trachea, adrenal gland andbone marrow. The rat tissues tested included: heart, brain, spleen,lung, liver, skeletal muscle, kidney, and testis.

There was a strong hybridization to human whole brain, the followinghuman brain regions: cerebellum, corpus callosum, cerebral cortex,medulla, occipital pole, frontal lobe, temporal lobe, putamen, amygdala,caudate nucleus, hippocampus, substantia nigra, subthalamic nucleus andthalamus; and rat brain indicating that the approximately 3 kb mACHR-6gene transcript is expressed in these tissues. There was alsohybridization to human spinal cord.

In Situ Hybridization

For in situ analysis, the brain of an adult Sprague-Dawley rat wasremoved and frozen on dry ice. Ten-micrometer-thick coronal sections ofthe brain were postfixed with 4% formaldehyde in DEPC treated 1×phosphate-buffered saline at room temperature for 10 minutes beforebeing rinsed twice in DEPC 1× phosphate-buffered saline and once in 0.1M triethanolamine-HCl (pH 8.0). Following incubation in 0.25% aceticanhydride-0.1 M triethanolamine-HCl for 10 minutes, sections were rinsedin DEPC 2×SSC (1×SSC is 0.15M NaCl plus 0.015M sodium citrate). Tissuewas then dehydrated through a series of ethanol washes, incubated in100% chloroform for 5 minutes, and then rinsed in 100% ethanol for 1minute and 95% ethanol for 1 minute and allowed to air dry.

Hybridizations were performed with ³⁵S-radiolabeled (5×10⁷ cpm/ml) cRNAprobes encoding a 474-bp fragment of the rat gene (generated with PCRprimers F, 5′-CAAGAACCCTTTAAGCCAAG (SEQ ID NO:27), and R,5′-GAAGAAGGTAACGCTGAGGA (SEQ ID NO:28)) and a 529-bp fragment of the ratgene (generated with PCR primers F, 5′-CAGAACCCCCACCAGATGCC (SEQ IDNO:29), and R, 5′-TAGTGGCACAGTGGGTAGAG (SEQ ID NO:30)). Probes wereincubated in the presence of a solution containing 600 mM NaCl, 10 mMTris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeasttRNA, 0.05% yeast total-RNA type X1, 1× Denhardt's solution, 50%formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodiumdodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hours at 55°C.

After hybridization, slides were washed with 2×SSC. Sections were thensequentially incubated at 37° C. in TNE (a solution containing 10 mMTris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNEwith 10 μg of RNase A per ml for 30 minutes, and finally in TNE for 10minutes. Slides were then rinsed with 2×SSC at room temperature, washedwith 2×SSC at 50° C. for 1 hour, washed with 0.2×SSC at 55° C. for 1hour, and 0.2×SSC at 60° C. for 1 hour. Sections were then dehydratedrapidly through serial ethanol-0.3 M sodium acetate concentrationsbefore being air dried and exposed to Kodak Biomax MR scientific imagingfilm for 24 hours and subsequently dipped in NB-2 photoemulsion andexposed at 4° C. for 7 days before being developed and counter stained.

Significant hybridization was seen in a number of brain regions. Theseincluded the cortex, caudate putamen, hippocampus, thalamus andcerebellum. Analysis of these regions at high magnification showed thatsignificant labeling was seen over the cell bodies of neurons.

Example 3 Expression of Recombinant mACHR-6 Polypeptide in BacterialCells

In this example, mACHR-6 is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, mACHR-6is fused to GST and this fusion polypeptide is expressed in E. coli,e.g., strain PEB 199. As the human and rat mACHR-6 polypeptides arepredicted to be approximately 51.3 kDa, and 51.2 kDa, respectively, andGST is predicted to be 26 kDa, the fusion polypeptides are predicted tobe approximately 77.3 kDa and 77.2 kDa, respectively, in molecularweight. Expression of the GST-mACHR-6 fusion polypeptide in PEB 199 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced PEB 199 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 4 Expression of Recombinant mACHR-6 Polypeptide in Cos Cells

To express the mACHR-6 gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire mACHR-6 polypeptide and a HA tag (Wilson et al. (1984) Cell37:767) fused in-frame to its 3′ end of the fragment is cloned into thepolylinker region of the vector, thereby placing the expression of therecombinant polypeptide under the control of the CMV promoter.

To construct the plasmid, the mACHR-6 DNA sequence is amplified by PCRusing two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the mACHR-6coding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag and the last 20nucleotides of the mACHR-6 coding sequence. The PCR amplified fragmentand the pCDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the mACHR-6 gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5a, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

COS cells are subsequently transfected with the mACHR-6-pcDNA/Ampplasmid DNA using the calcium phosphate or calcium chlorideco-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning. A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The expression of the mACHR-6 polypeptide is detected byradiolabelling (³⁵S-methionine or ³⁵S-cysteine available from NEN,Boston, Mass., can be used) and immunoprecipitation (Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonalantibody. Briefly, the cells are labelled for 8 hours with³⁵S-methionine (or ³⁵S-cysteine). The culture media are then collectedand the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1%NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate andthe culture media are precipitated with an HA specific monoclonalantibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the mACHR-6 coding sequence is cloneddirectly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of themACHR-6 polypeptide is detected by radiolabelling andimmunoprecipitation using an mACHR-6 specific monoclonal antibody.

Example 5 Characterization of the Human and Rat mACHR-6 Polypeptides

In this example, the amino acid sequences of the human and the ratmACHR-6 polypeptides were compared to amino acid sequences of knownpolypeptides and various motifs were identified.

The human mACHR-6 polypeptide, the amino acid sequence of which is shownin FIG. 1 (SEQ ID NO:2), is a novel polypeptide which includes 445 aminoacid residues. The human mACHR-6 polypeptide contains seventransmembrane domains between amino acid residues 34-59 (SEQ ID NO:7),73-91 (SEQ ID NO:8), 109-130 (SEQ ID NO:9), 152-174 (SEQ ID NO:10),197-219 (SEQ ID NO:11), 360-380 (SEQ ID NO:12), and 396-416 (SEQ IDNO:13). The nucleotide sequence of the human mACHR-6 was used as adatabase query using the BLASTN program (BLASTN1.3MP, Altschul et al.(1990) J. Mol. Biol. 215:403). The closest hits were human, rat, mouseand pig mACHR M1 (GenBank™ Accession Numbers P11229, P08482, P12657, andP04761, respectively). The highest similarity is 32/70 amino acididentities.

The rat mACHR-6 polypeptide, the amino acid sequence of which is shownin FIG. 2 (SEQ ID NO:5), is a novel polypeptide which includes 445 aminoacid residues. The rat mACHR-6 polypeptide contains seven transmembranedomains between amino acid residues 34-59 (SEQ ID NO:14), 73-91 (SEQ IDNO:15), 109-130 (SEQ ID NO:16), 152-174 (SEQ ID NO:17), 197-219 (SEQ IDNO:18),360-380 (SEQ ID NO:19) and 396-416 (SEQ ID NO:20), whichcorrespond to the human mACHR-6 polypeptide transmembrane domains 1-7(SEQ ID NOs:7-13). The nucleotide sequence of the rat mACHR-6 was usedas a database query using the BLASTN program (BLASTN1.3MP, Altschul etal. (1990) J. Mol. Biol. 215:403). The closest hits were human, rat,mouse and pig mACHR M1 (GenBank™ Accession Numbers P11229, P08482,P12657, and P04761, respectively). The highest similarity is 33/70 aminoacid identities. Hydropathy plots indicated that the transmembranedomains of the rat mACHR-6 polypeptide are similar to those of the ratmACHR M1. The cysteines (residues 63 and 44 of SEQ ID NO:5) that giverise to intramolecular disulfide bonds are also conserved.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A non-human transgenic animal, which expresses an mACHR-6 polypeptideor a biologically active portion thereof.
 2. The transgenic animal ofclaim 1, wherein said mACHR-6 polypeptide is a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 2, 5 or
 32. 3. The transgenicanimal of claim 1, wherein said animal exhibits a central nervous systemdisorder.
 4. The transgenic animal of claim 1, wherein said animalexhibits a disorder selected from the group consisting of a cognitivedisorder and an eating disorder.
 5. A non-human transgenic animalcontaining a transgene comprising the nucleotide sequence of SEQ IDNO:1, 4, or 31, or a fragment thereof.
 6. The transgenic animal of claim5, wherein the transgene comprises intronic sequences or polyadenylationsignals.
 7. The transgenic animal of claim 5, wherein the transgene isoperably linked to a tissue-specific regulatory sequence.
 8. Thetransgenic animal of claim 5, wherein said animal exhibits a cognitivedisorder.
 9. The transgenic animal of claim 8, wherein said cognitivedisorder is selected from the group consisting of a memory disorder, alearning disorder, a learning disability, and Alzheimer's related memoryloss.
 10. The transgenic animal of claim 5, wherein said animal exhibitsan eating disorder.
 11. The transgenic animal of claim 5, wherein saidanimal is homozygous or heterozygous for said transgene.
 12. Thetransgenic animal of claim 5, wherein said animal is a mouse.
 13. Anisolated cell, or a purified preparation of cells from the transgenicanimal of claim
 5. 14. A homologous recombinant mouse in which the geneencoding the GPCR mACHR-6 is overexpressed.
 15. The homologousrecombinant mouse of claim 14, wherein said mouse exhibits an eatingdisorder.
 16. The homologous recombinant mouse of claim 14, wherein saidmouse exhibits a cognitive disorder.
 17. The homologous recombinantmouse of claim 16, wherein said cognitive disorder is selected from thegroup consisting of a memory disorder, a learning disorder, a learningdisability, and Alzheimer's related memory loss.
 18. An isolated cell,or a purified preparation of cells from the homologous recombinant mouseof claim
 14. 19. The isolated cell of claim 18, wherein the cell isselected from the group consisting of a brain cell, a gland cell and asmooth muscle cell.
 20. The isolated cell of claim 18, wherein the cellis a neurotransmitter responsive cell.